Anti-c5 antibodies having improved pharmacokinetics

ABSTRACT

The disclosure provides antibodies that are useful for, among other things, inhibiting terminal complement (e.g., the assembly and/or activity of the C5b-9 TCC) and C5a anaphylatoxin-mediated inflammation and, thus, treating complement-associated disorders. The antibodies have a number of improved properties relative to eculizumab, including, e.g., increased serum half-life in a human.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/160,364 (filed May 20, 2016), which is a continuation of U.S. patentapplication Ser. No. 14/923,879 (filed Oct. 27, 2015, now U.S. Pat. No.9,371,377), which is a continuation of U.S. patent application Ser. No.14/789,329 (filed Jul. 1, 2015, now U.S. Pat. No. 9,206,251), which is adivisional of U.S. patent application Ser. No. 14/727,313 (filed Jun. 1,2015, now U.S. Pat. No. 9,107,861), which is a divisional of U.S. patentapplication Ser. No. 14/641,026 (filed on Mar. 6, 2015, now U.S. Pat.No. 9,079,949), which claims priority to and the benefit of U.S.provisional patent application No. 61/949,932 (filed on Mar. 7, 2014),the disclosures of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 20, 2017, isnamed AXJ_186DV2CN3_SL.txt and is 67,353 bytes in size.

TECHNICAL FIELD

The field of the invention is medicine, immunology, molecular biology,and protein chemistry.

BACKGROUND

The complement system acts in conjunction with other immunologicalsystems of the body to defend against intrusion of cellular and viralpathogens. There are at least 25 complement proteins, which are found asa complex collection of plasma proteins and membrane cofactors. Theplasma proteins make up about 10% of the globulins in vertebrate serum.Complement components achieve their immune defensive functions byinteracting in a series of intricate but precise enzymatic cleavage andmembrane binding events. The resulting complement cascade leads to theproduction of products with opsonic, immunoregulatory, and lyticfunctions. A concise summary of the biologic activities associated withcomplement activation is provided, for example, in The Merck Manual,16^(th) Edition.

The complement cascade can progress via the classical pathway (CP), thelectin pathway, or the alternative pathway (AP). The lectin pathway istypically initiated with binding of mannose-binding lectin (MBL) to highmannose substrates. The AP can be antibody independent, and can beinitiated by certain molecules on pathogen surfaces. The CP is typicallyinitiated by antibody recognition of, and binding to, an antigenic siteon a target cell. These pathways converge at the C3 convertase—the pointwhere complement component C3 is cleaved by an active protease to yieldC3a and C3b.

The AP C3 convertase is initiated by the spontaneous hydrolysis ofcomplement component C3, which is abundant in the plasma fraction ofblood. This process, also known as “tickover,” occurs through thespontaneous cleavage of a thioester bond in C3 to form C3i or C3(H₂O).Tickover is facilitated by the presence of surfaces that support thebinding of activated C3 and/or have neutral or positive chargecharacteristics (e.g., bacterial cell surfaces). This formation ofC3(H₂O) allows for the binding of plasma protein Factor B, which in turnallows Factor D to cleave Factor B into Ba and Bb. The Bb fragmentremains bound to C3 to form a complex containing C3(H₂O)Bb—the“fluid-phase” or “initiation” C3 convertase. Although only produced insmall amounts, the fluid-phase C3 convertase can cleave multiple C3proteins into C3a and C3b and results in the generation of C3b and itssubsequent covalent binding to a surface (e.g., a bacterial surface).Factor B bound to the surface-bound C3b is cleaved by Factor D to thusform the surface-bound AP C3 convertase complex containing C3b,Bb. (See,e.g., Müller-Eberhard (1988) Ann Rev Biochem 57:321-347.)

The AP C5 convertase—(C3b)₂,Bb—is formed upon addition of a second C3bmonomer to the AP C3 convertase. (See, e.g., Medicus et al. (1976) J ExpMed 144:1076-1093 and Fearon et al. (1975) J Exp Med 142:856-863.) Therole of the second C3b molecule is to bind C5 and present it forcleavage by Bb. (See, e.g., Isenman et al. (1980) J Immunol124:326-331.) The AP C3 and C5 convertases are stabilized by theaddition of the trimeric protein properdin as described in, e.g.,Medicus et al. (1976), supra. However, properdin binding is not requiredto form a functioning alternative pathway C3 or C5 convertase. (See,e.g., Schreiber et al. (1978) Proc Natl Acad Sci USA 75: 3948-3952 andSissons et al. (1980) Proc Natl Acad Sci USA 77: 559-562).

The CP C3 convertase is formed upon interaction of complement componentC1, which is a complex of C1q, C1r, and C1s, with an antibody that isbound to a target antigen (e.g., a microbial antigen). The binding ofthe C1q portion of C1 to the antibody-antigen complex causes aconformational change in C1 that activates C1r. Active C1r then cleavesthe C1-associated C1s to thereby generate an active serine protease.Active C1s cleaves complement component C4 into C4b and C4a. Like C3b,the newly generated C4b fragment contains a highly reactive thiol thatreadily forms amide or ester bonds with suitable molecules on a targetsurface (e.g., a microbial cell surface). C1s also cleaves complementcomponent C2 into C2b and C2a. The complex formed by C4b and C2a is theCP C3 convertase, which is capable of processing C3 into C3a and C3b.The CP C5 convertase—C4b,C2a,C3b—is formed upon addition of a C3bmonomer to the CP C3 convertase. (See, e.g., Müller-Eberhard (1988),supra and Cooper et al. (1970) J Exp Med 132:775-793.)

In addition to its role in C3 and C5 convertases, C3b also functions asan opsonin through its interaction with complement receptors present onthe surfaces of antigen-presenting cells such as macrophages anddendritic cells. The opsonic function of C3b is generally considered tobe one of the most important anti-infective functions of the complementsystem. Patients with genetic lesions that block C3b function are proneto infection by a broad variety of pathogenic organisms, while patientswith lesions later in the complement cascade sequence, i.e., patientswith lesions that block C5 functions, are found to be more prone only toNeisseria infection, and then only somewhat more prone.

The AP and CP C5 convertases cleave C5 into C5a and C5b. Cleavage of C5releases C5a, a potent anaphylatoxin and chemotactic factor, and C5b,which allows for the formation of the lytic terminal complement complex,C5b-9. C5b combines with C6, C7, and C8 to form the C5b-8 complex at thesurface of the target cell. Upon binding of several C9 molecules, themembrane attack complex (MAC, C5b-9, terminal complement complex—TCC) isformed. When sufficient numbers of MACs insert into target cellmembranes the openings they create (MAC pores) mediate rapid osmoticlysis of the target cells.

While a properly functioning complement system provides a robust defenseagainst infecting microbes, inappropriate regulation or activation ofthe complement pathways has been implicated in the pathogenesis of avariety of disorders including, e.g., rheumatoid arthritis (RA); lupusnephritis; asthma; ischemia-reperfusion injury; atypical hemolyticuremic syndrome (aHUS); dense deposit disease (DDD); paroxysmalnocturnal hemoglobinuria (PNH); macular degeneration (e.g., age-relatedmacular degeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP);spontaneous fetal loss; Pauci-immune vasculitis; epidermolysis bullosa;recurrent fetal loss; multiple sclerosis (MS); traumatic brain injury;and injury resulting from myocardial infarction, cardiopulmonary bypassand hemodialysis. (See, e.g., Holers et al. (2008) Immunological Reviews223:300-316.) The down-regulation of complement activation has beendemonstrated to be effective in treating several disease indications ina variety of animal models. See, e.g., Rother t al. (2007) NatureBiotechnology 25(11):1256-1264; Wang et al. (1996) Proc Natl Acad SciUSA 93:8563-8568; Wang et al. (1995) Proc Natl Acad Sci USA92:8955-8959; Rinder et al. (1995) J Clin Invest 96:1564-1572; Kroshuset al. (1995) Transplantation 60:1194-1202; Homeister et al. (1993) JImmunol 150:1055-1064; Weisman et al. (1990) Science 249:146-151;Amsterdam et al. (1995) Am J Physiol 268:H448-H457; and Rabinovici etal. (1992) J Immunol 149:1744 1750.

SUMMARY

The disclosure relates to anti-C5 antibodies that have one of moreimproved characteristics, e.g., relative to known anti-C5 antibodiesused for therapeutic purposes. For example, the anti-C5 antibodiesdescribed herein exhibit increased serum-life relative to the serumelimination half-life of eculizumab. Because of their improvedpharmacokinetic properties, the antibodies described herein feature anumber of advantages, e.g., advantages over other anti-C5 antibodiesthat bind to, and inhibit cleavage of, full-length or mature C5. Likesuch anti-C5 antibodies, the antibodies described herein can inhibit theC5a-mediated inflammatory response and the C5b (MAC)-dependent celllysis that results from cleavage of C5. However, as the concentration ofC5 in human plasma is approximately 0.37 μM (Rawal and Pangburn (2001) JImmunol 166(4):2635-2642), the use of high concentrations and/orfrequent administration of anti-C5 antibodies, such as eculizumab, isoften necessary to effectively inhibit C5 in a human. The disclosuresets forth in the working examples experimental data evidencing thatwhile anti-C5 antibodies are highly effective at inhibiting complementin vitro and in vivo (see, e.g., Hillmen et al. (2004) N Engl J Med350(6):552), the antibodies are particularly susceptible totarget-mediated clearance because of the high concentration of C5 inblood. The disclosure also shows that the new antibodies describedherein have reduced susceptibility to the target-mediated clearance andthus have a longer serum elimination half-life (half-life), as comparedto previously known anti-C5 antibodies, in blood.

In view of their longer half-life, the antibodies described herein canbe administered to a human at a much lower dose and/or less frequentlythan previously known anti-C5 antibodies (such as, eculizumab) andeffectively provide the same or greater inhibition of C5 in a human. Theability to administer a lower dose of the antibody, as compared to,e.g., the dose of eculizumab, also allows for additional delivery routessuch as, e.g., subcutaneous administration, intramuscularadministration, intrapulmonary delivery, and administration via the useof biologically degradable microspheres.

Accordingly, in one aspect, the disclosure features an anti-C5 antibodyhaving one or more improved properties (e.g., improved pharmacokineticproperties) relative to eculizumab. The antibody or C5-binding fragmentthereof is one that: (a) binds to complement component C5; (b) inhibitsthe cleavage of C5 into fragments C5a and C5b; and (c) comprises: (i) aheavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:1, (ii) a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:2, (iii) a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:3, (iv) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:4, (v) a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:5,and (vi) a light chain CDR3 comprising the amino acid sequence depictedin SEQ ID NO:6, in which at least one (e.g., at least two, at leastthree, at least four, at least five, at least six, at least seven, or atleast eight) amino acid(s) of (i)-(vi) is substituted with a differentamino acid. In some embodiments, the C5 is human C5.

In some embodiments of any of the antibodies or fragments describedherein, at least one amino acid of heavy chain CDR1 is substituted witha different amino acid. In some embodiments of any of the antibodies orfragments described herein, at least one amino acid of heavy chain CDR2is substituted with a different amino acid. In some embodiments of anyof the antibodies or fragments described herein, at least one amino acidof heavy chain CDR3 is substituted with a different amino acid.

In some embodiments of any of the antibodies or fragments describedherein at least one amino acid of light chain CDR1 is substituted with adifferent amino acid. In some embodiments of any of the antibodies orfragments described herein, the glycine at position 8 relative to SEQ IDNO:4 is substituted with a different amino acid (e.g., a histidine).

In some embodiments of any of the antibodies or fragments describedherein, at least one amino acid of light chain CDR2 is substituted witha different amino acid. In some embodiments of any of the antibodies orfragments described herein, at least one amino acid of light chain CDR3is substituted with a different amino acid.

In some embodiments of any of the antibodies or fragments describedherein, a substitution is made at an amino acid position selected fromthe group consisting of: glycine at position 1 relative to SEQ ID NO:1,tyrosine at position 2 relative to SEQ ID NO:1, isoleucine at position 3relative to SEQ ID NO:1, phenylalanine at position 4 relative to SEQ IDNO:1, serine at position 5 relative to SEQ ID NO:1, asparagine atposition 6 relative to SEQ ID NO:1, tyrosine at position 7 relative toSEQ ID NO:1, tryptophan at position 8 relative to SEQ ID NO:1,isoleucine at position 9 relative to SEQ ID NO:1, glutamine at position10 relative to SEQ ID NO:1, glutamic acid at position 1 relative to SEQID NO:2, isoleucine at position 2 relative to SEQ ID NO:2, leucine atposition 3 relative to SEQ ID NO:2, proline at position 4 relative toSEQ ID NO:2, glycine at position 5 relative to SEQ ID NO:2, serine atposition 6 relative to SEQ ID NO:2, glycine at position 7 relative toSEQ ID NO:2, serine at position 8 relative to SEQ ID NO:2, threonine atposition 9 relative to SEQ ID NO:2, glutamic acid at position 10relative to SEQ ID NO:2, tyrosine at position 11 relative to SEQ IDNO:2, threonine at position 12 relative to SEQ ID NO:2, glutamic acid atposition 13 relative to SEQ ID NO:2, asparagine at position 14 relativeto SEQ ID NO:2, phenylalanine at position 15 relative to SEQ ID NO:2,lysine at position 16 relative to SEQ ID NO:2, aspartic acid at position17 relative to SEQ ID NO:2, tyrosine at position 1 relative to SEQ IDNO:3, phenylalanine at position 2 relative to SEQ ID NO:3, phenylalanineat position 3 relative to SEQ ID NO:3, glycine at position 4 relative toSEQ ID NO:3, serine at position 5 relative to SEQ ID NO:3, serine atposition 6 relative to SEQ ID NO:3, proline at position 7 relative toSEQ ID NO:3, asparagine at position 8 relative to SEQ ID NO:3,tryptophan at position 9 relative to SEQ ID NO:3, tyrosine at position10 relative to SEQ ID NO:3, phenylalanine at position 11 relative to SEQID NO:3, aspartic acid at position 12 relative to SEQ ID NO:3, andvaline at position 13 relative to SEQ ID NO:3.

In some embodiments of any of the antibodies or fragments describedherein, a substitution is made at an amino acid position selected fromthe group consisting of: glycine at position 8 relative to SEQ ID NO:4,leucine at position 10 relative to SEQ ID NO:4, valine at position 3relative to SEQ ID NO:6, and threonine at position 6 relative to SEQ IDNO:6.

In some embodiments of any of the antibodies or fragments describedherein, a substitution is made at an amino acid position selected fromthe group consisting of: tyrosine at position 2 relative to SEQ ID NO:1,isoleucine at position 9 relative to SEQ ID NO:1, leucine at position 3relative to SEQ ID NO:2, and serine at position 8 relative to SEQ IDNO:2.

In some embodiments of any of the antibodies or fragments describedherein, both tyrosine at position 2 relative to SEQ ID NO:1 and leucineat position 3 relative to SEQ ID NO:2 are substituted with a differentamino acid. In some embodiments of any of the antibodies or fragmentsdescribed herein, the different amino acid is a histidine.

In some embodiments of any of the antibodies or fragments describedherein, both isoleucine at position 9 relative to SEQ ID NO:1 and serineat position 8 relative to SEQ ID NO:2 are substituted with a differentamino acid. In some embodiments of any of the antibodies or fragmentsdescribed herein, both isoleucine at position 9 relative to SEQ ID NO:1and leucine at position 3 relative to SEQ ID NO:2 are substituted with adifferent amino acid. In some embodiments of any of the antibodies orfragments described herein, the different amino acid is a histidine.

In some embodiments of any of the antibodies or fragments describedherein, both tyrosine at position 2 relative to SEQ ID NO:1 and serineat position 8 relative to SEQ ID NO:2 are substituted with a differentamino acid. In some embodiments of any of the antibodies or fragmentsdescribed herein, the antibody or antigen-binding fragment comprises acombination of amino acid substitutions selected from Table 1. In someembodiments of any of the antibodies or fragments described herein, thedifferent amino acid is a histidine.

In some embodiments of any of the antibodies or fragments describedherein, the combination of amino acid substitutions comprises: (i) asubstitution of a different amino acid for glycine at position 8relative to SEQ ID NO:4 in the light chain polypeptide of the antibodyor antigen-binding fragment thereof; (ii) a substitution of a differentamino acid for glycine at position 2 relative to SEQ ID NO:1 of theheavy chain polypeptide of the antibody or antigen-binding fragmentthereof; and (iii) a substitution of a different amino acid for serineat position 8 relative to SEQ ID NO:2 of the heavy chain polypeptide ofthe antibody or antigen-binding fragment thereof. In some embodiments ofany of the antibodies or fragments described herein, the different aminoacid is a histidine.

In some embodiments of any of the antibodies or fragments describedherein, tyrosine at position 2 relative to SEQ ID NO:1 and serine atposition 8 relative to SEQID NO:2 are substituted with histidine. Insome embodiments of any of the antibodies or fragments described herein,the different amino acid is a histidine.

In some embodiments, any of the antibodies or fragments described hereinbind to C5 at pH 7.4 and 25° C. with an affinity dissociation constant(K_(D)) that is in the range 0.1 nM≦K_(D)≦1 nM. In some embodiments, anyof the antibodies or fragments described herein bind to C5 at pH 7.4 and25° C. with a K_(D) that is in the range 0.2 nM≦K_(D)≦1 nM. In someembodiments, any of the antibodies or fragments described herein bind toC5 at pH 7.4 and 25° C. with a K_(D) that is in the range 0.5 nM≦K_(D)≦1nM.

In some embodiments, any of the antibodies or fragments described hereinbind to C5 at pH 6.0 and 25° C. with a K_(D) that is ≧1 nM (e.g., ≧50nM, ≧100 nM, or ≧1 μM).

In some embodiments of any of the antibodies or fragments describedherein, the [(K_(D) of the antibody or antigen-binding fragment thereoffor C5 at pH 6.0 and at 25° C.)/(K_(D) of the antibody orantigen-binding fragment thereof for C5 at pH 7.4 and at 25° C.)] isgreater than 25. In some embodiments of any of the antibodies orfragments described herein, the [(K_(D) of the antibody orantigen-binding fragment thereof for C5 at pH 6.0 and at 25° C.)/(K_(D)of the antibody or antigen-binding fragment thereof for C5 at pH 7.4 andat 25° C.)] is greater than 100 (e.g., greater than 200, 300, 400, 500,600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, or8500).

In some embodiments of any of the antibodies or fragments describedherein, the K_(D) of the antibody or antigen-binding fragment thereoffor C5 at pH 7.4 and at 25° C. is less than 1 (e.g., less than 0.9, 0.8,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) nM.

Based on the characterization of several variant eculizumab molecules asdescribed in the working examples, the inventors discovered a new genusof antibodies having improved pharmacokinetic properties as compared toeculizumab. Antibodies within this genus have an affinity for C5 that isweaker than the affinity of eculizumab for C5 at pH 7.4. Yet theantibodies have an affinity dissociation constant (K_(D)) for C5 at pH7.4 that is equal to or less than 1 nM. While the disclosure is notbound by any particular theory or mechanism of action, the inventorsbelieve that slightly reducing the affinity of eculizumab for C5 at pH7.4, and its subsequent effect on the affinity of the antibody for C5 atpH 6.0 while maintaining the same/similar ratio of affinity at pH7.4 andpH 6.0, will substantially reduce the C5-mediated clearance of theantibody without substantially affecting the complement inhibitoryfunction of the resultant antibody in patients. Thus, the inventors havedefined an optimal affinity range for anti-C5 antibodies giving rise toimproved pharmacokinetic properties while preserving the requiredpharmacodynamic properties, each relative to eculizumab. Accordingly, inanother aspect, the disclosure features an isolated antibody, orantigen-binding fragment thereof, that: (a) binds to complementcomponent C5 at pH 7.4 and 25° C. with an affinity dissociation constant(K_(D)) that is ≦1 nM; (b) binds to C5 at pH 6.0 and 25° C. with a K_(D)that is no lower than 10 nM; (c) inhibits the cleavage of C5 intofragments C5a and C5b, wherein the [(K_(D) of the antibody orantigen-binding fragment thereof for C5 at pH 6.0 and 25° C.)/(K_(D) ofthe antibody or antigen-binding fragment thereof for C5 at pH 7.4 and25° C.)] is greater than or equal to 25.

In some embodiments, the antibody or antigen-binding fragment thereofbinds to C5 at pH 7.4 and 25° C. with an affinity dissociation constant(K_(D)) that is in the range 0.1 nM≦K_(D)≦1 nM. In some embodiments, theantibody or antigen-binding fragment thereof binds to C5 at pH 7.4 and25° C. with a K_(D) that is in the range 0.2 nM≦K_(D)≦1 nM. In someembodiments, the antibody or antigen-binding fragment thereof binds toC5 at pH 7.4 and 25° C. with a K_(D) that is in the range 0.5 nM≦K_(D)≦1nM. In some embodiments, the antibody or antigen-binding fragmentthereof binds to C5 at pH 6.0 and 25° C. with a K_(D) that is ≧1 nM. Insome embodiments, the antibody or antigen-binding fragment thereof bindsto C5 at pH 6.0 and 25° C. with a K_(D) that is ≧50 nM. In someembodiments, the antibody or antigen-binding fragment thereof binds toC5 at pH 6.0 and 25° C. with a K_(D) that is ≧100 nM. In someembodiments, the antibody or antigen-binding fragment thereof binds toC5 at pH 6.0 and 25° C. with a K_(D) that is ≧1 μM.

In some embodiments, the [(K_(D) of the antibody or antigen-bindingfragment thereof for C5 at pH 6.0 and at 25° C.)/(K_(D) of the antibodyor antigen-binding fragment thereof for C5 at pH 7.4 and at 25° C.)] isgreater than 50 (e.g., greater than 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500,7000, 7500, 8000, or 8500).

In some embodiments, the antibody or antigen-binding fragment thereofbinds to C5 at pH 7.4 and at 25° C. with a K_(D)<1 nM. In someembodiments, the antibody or antigen-binding fragment thereof binds toC5 at pH 7.4 and at 25° C. with a K_(D)<0.8 nM. In some embodiments, theantibody or antigen-binding fragment thereof binds to C5 at pH 7.4 andat 25° C. with a K_(D)<0.5 nM. In some embodiments, the antibody orantigen-binding fragment thereof binds to C5 at pH 7.4 and at 25° C.with a K_(D)<0.2 nM.

In some embodiments, the antibody or antigen-binding fragment thereofcomprises: (i) a heavy chain CDR1 comprising the amino acid sequencedepicted in SEQ ID NO:1, (ii) a heavy chain CDR2 comprising the aminoacid sequence depicted in SEQ ID NO:2, (iii) a heavy chain CDR3comprising the amino acid sequence depicted in SEQ ID NO:3, (iv) a lightchain CDR1 comprising the amino acid sequence depicted in SEQ ID NO:4,(v) a light chain CDR2 comprising the amino acid sequence depicted inSEQ ID NO:5, and (vi) a light chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:6, in which at least one amino acid of(i)-(vi) is substituted with a different amino acid. The different aminoacid can be any amino acid (e.g., a histidine). In some embodiments, atleast one amino acid of heavy chain CDR1 is substituted with a differentamino acid. In some embodiments, at least one amino acid of heavy chainCDR2 is substituted with a different amino acid. In some embodiments, atleast one amino acid of heavy chain CDR3 is substituted with a differentamino acid. In some embodiments, at least one amino acid of light chainCDR1 is substituted with a different amino acid. In some embodiments, atleast one amino acid of light chain CDR2 is substituted with a differentamino acid. In some embodiments, at least one amino acid of light chainCDR3 is substituted with a different amino acid.

In some embodiments, a substitution is made at an amino acid positionselected from the group consisting of: glycine at position 8 relative toSEQ ID NO:4, leucine at position 10 relative to SEQ ID NO:4, valine atposition 3 relative to SEQ ID NO:6, and threonine at position 6 relativeto SEQ ID NO:6. In some embodiments, a substitution is made at an aminoacid position selected from the group consisting of: tyrosine atposition 2 relative to SEQ ID NO:1, isoleucine at position 9 relative toSEQ ID NO:1, leucine at position 3 relative to SEQ ID NO:2, and serineat position 8 relative to SEQ ID NO:2. In some embodiments, the antibodyor antigen-binding fragment comprises a combination of amino acidsubstitutions selected from Table 1.

In some embodiments, a combination of amino acid substitutionsintroduced into the CDRs comprises: (i) a substitution a different aminoacid for glycine at position 8 relative to SEQ ID NO:4 in the lightchain polypeptide of the antibody or antigen-binding fragment thereof;(ii) a substitution of a different amino acid for glycine at position 2relative to SEQ ID NO:1 of the heavy chain polypeptide of the antibodyor antigen-binding fragment thereof; and (iii) a substitution of adifferent amino acid for serine at position 8 relative to SEQ ID NO:2 ofthe heavy chain polypeptide of the antibody or antigen-binding fragmentthereof.

In some embodiments, a combination of amino acid substitutionscomprises: (i) a substitution of a different amino acid for glycine atposition 2 relative to SEQ ID NO:1 of the heavy chain polypeptide of theantibody or antigen-binding fragment thereof; and (ii) a substitution ofa different amino acid for serine at position 8 relative to SEQ ID NO:2of the heavy chain polypeptide of the antibody or antigen-bindingfragment thereof.

In some embodiments, tyrosine at position 2 relative to SEQ ID NO:1 andserine at position 8 relative to SEQID NO:2 are substituted (e.g., withhistidine).

In some embodiments, any of the antibodies or fragment thereof comprisea variant human Fc constant region (e.g., a variant human IgG Fcconstant region) that binds to human neonatal Fc receptor (FcRn) withgreater affinity than that of the native human Fc constant region fromwhich the variant human Fc constant region was derived. The variant Fcconstant region can comprise one or more (e.g., two, three, four, orfive or more) amino acid substitutions relative to the native human Fcconstant region from which the variant human Fc constant region wasderived. The substitution can be at, e.g., amino acid position 237, 238,239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297,298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334,360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, or 436(EU numbering) relative to the native human Fc constant region. Thesubstitution can be one selected from the group consisting of:methionine for glycine at position 237; alanine for proline at position238; lysine for serine at position 239; isoleucine for lysine atposition 248; alanine, phenylalanine, isoleucine, methionine, glutamine,serine, valine, tryptophan, or tyrosine for threonine at position 250;phenylalanine, tryptophan, or tyrosine for methionine at position 252;threonine for serine at position 254; glutamic acid for arginine atposition 255; aspartic acid, glutamic acid, or glutamine for threonineat position 256; alanine, glycine, isoleucine, leucine, methionine,asparagine, serine, threonine, or valine for proline at position 257;histidine for glutamic acid at position 258; alanine for aspartic acidat position 265; phenylalanine for aspartic acid at position 270;alanine, or glutamic acid for asparagine at position 286; histidine forthreonine at position 289; alanine for asparagine at position 297;glycine for serine at position 298; alanine for valine at position 303;alanine for valine at position 305; alanine, aspartic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, valine,tryptophan, or tyrosine for threonine at position 307; alanine,phenylalanine, isoleucine, leucine, methionine, proline, glutamine, orthreonine for valine at position 308; alanine, aspartic acid, glutamicacid, proline, or arginine for leucine or valine at position 309;alanine, histidine, or isoleucine for glutamine at position 311;alanine, or histidine for aspartic acid at position 312; lysine, orarginine for leucine at position 314; alanine, or histidine forasparagine at position 315; alanine for lysine at position 317; glycinefor asparagine at position 325; valine for isoleucine at position 332;leucine for lysine at position 334; histidine for lysine at position360; alanine for aspartic acid at position 376; alanine for glutamicacid at position 380; alanine for glutamic acid at position 382; alaninefor asparagine or serine at position 384; aspartic acid, or histidinefor glycine at position 385; proline for glutamine at position 386;glutamic acid for proline at position 387; alanine, or serine forasparagine at position 389; alanine for serine at position 424; alanine,aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, asparagine, proline, glutamine, serine, threonine, valine,tryptophan, or tyrosine for methionine at position 428; lysine forhistidine at position 433; alanine, phenylalanine, histidine, serine,tryptophan, or tyrosine for asparagine at position 434;

and histidine for tyrosine or phenylalanine at position 436, all in EUnumbering.

In some embodiments of any of the antibodies or antigen-bindingfragments described herein, the variant human Fc constant regioncomprises a methionine at position 428 and an asparagine at position434, each in EU numbering.

In some embodiments, any of the antibodies or antigen-binding fragmentsthereof can comprise, or consist of, a heavy chain polypeptidecomprising the amino acid sequence depicted in SEQ ID NO:12 or 14 and alight chain polypeptide comprising the amino acid sequence depicted inSEQ ID NO:8 or 11.

The disclosure also features an antibody comprising the heavy chainvariable region of eculizumab (SEQ ID NO:7) or the CDRs of the heavychain region of eculizumab (SEQ ID NOs:1-3) and any of the variant humanFc constant regions described herein, e.g., the variant human Fcconstant region comprising a methionine at position 428 and anasparagine at position 434, each in EU numbering.

In one embodiment, the antibody or antigen binding fragment has anincreased half-life in humans relative to half-life in serum ofeculizumab. The half-life as used herein is defined as the time it takesfor the plasma concentration of the antibody drug in the body to bereduced by one half or 50%. This 50% reduction in serum concentrationreflects the amount of drug circulating and not removed by the naturalmethods of antibody clearance. The half-life of eculizumab has beendetermined to be 272+82 hours or 11.3 days in PNH patients and 12.1 daysin aHUS patients (See Soliris Prescribing information). The half-life inhumans of antibodies or fragments described herein may be increasedrelative to the half-life in humans of eculizumab. The half-lifeincrease relative to eculizumab may be at least 1.5 times the half lifeeculizumab, at least 2 times the half life eculizumab, at least 2.5times the half-life of eculizumab or at least 3 times the half-life ofeculizumab.

In some embodiments of any of the antibodies or fragments describedherein, the antibody has a serum half-life in a human that is greaterthan, or at least, 10 (e.g., greater than, or at least, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39 or 40) days. This half-life (or extension ofhalf-life relative to eculizumab) can, in some embodiments, be achievedby an antibody described herein containing a naturally-occurring humanFc constant region. In some embodiments, the half-life is measuredrelative to an antibody comprising a variant human Fc constant regiondescribed herein. The half-life in humans of antibodies or fragmentsdescribed herein may be increased relative to the half-life in humans ofeculizumab. The half-life in humans of the antibody described herein isat least 25 days, at least 26 days, at least 27 days, at least 28 days,at least 29 days, at least 30 days, at least 31 days, at least 32 days,at least 33 days, at least 34 days, or at least 35 days.

In some embodiments, any of the antibodies or fragments described hereinare humanized, fully human, deimmunized, or chimeric. In someembodiments, an antibody or fragment thereof described herein can be,e.g., a recombinant antibody, a single chain antibody, a diabody, anintrabody, an Fv fragment, an Fd fragment, an Fab fragment, an Fab′fragment, and an F(ab′)₂ fragment.

In some embodiments, any of the antibodies or fragments thereofdescribed herein can comprise a heterologous moiety, e.g., a sugar. Forexample, the antibody or fragment thereof can be glycosylated. Theheterologous moiety can also be a detectable label, e.g., a fluorescentlabel, a luminescent label, a heavy metal label, a radioactive label, oran enzymatic label.

In some embodiments, any of the antibodies or antigen-binding fragmentsthereof described herein can be manufactured in a CHO cell. In someembodiments, the antibodies or antigen-binding fragments thereof do notcontain detectable sialic acid residues.

In some embodiments, any of the antibodies or antigen-binding fragmentsthereof described herein can be modified with a moiety that improves oneor both of: (a) the stabilization of the antibody or antigen-bindingfragment thereof in circulation and (b) the retention of the antibody orantigen-binding fragment thereof in circulation. Such a moiety can bePEG (PEGylation).

In yet another aspect, the disclosure features a nucleic acid thatencodes one or both of the heavy and light chain polypeptides of any ofthe antibodies or antigen-binding fragments described herein. Alsofeatured is a vector (e.g., a cloning or expression vector) comprisingthe nucleic acid and a cell (e.g., an insect cell, bacterial cell,fungal cell, or mammalian cell) comprising the vector. The disclosurefurther provides a method for producing any of the antibodies orantigen-binding fragments thereof described herein. The methods include,optionally, providing the above described cell (or culture of cells)containing an expression vector (integrated or extrachromosomal), whichvector contains a nucleic acid that encodes one or both of the heavy andlight chain polypeptides of any of the antibodies or antigen-bindingfragments described herein. The cell or culture of cells is culturedunder conditions and for a time sufficient to allow expression by thecell (or culture of cells) of the antibody or antigen-binding fragmentthereof encoded by the nucleic acid. The method can also includeisolating the antibody or antigen-binding fragment thereof from the cell(or cells of the culture) or from the media in which the cell or cellswere cultured.

In another aspect, the disclosure features a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and one or more of anyof the antibodies or antigen-binding fragments thereof described herein.

In another aspect, the disclosure features a therapeutic kit comprising:(i) one or more of any of the antibodies or antigen-binding fragmentsthereof described herein and (ii) means for delivery of the antibody orantigen-binding fragment thereof to a human. The means can be, e.g., asyringe or a pump.

In yet another aspect, the disclosure features an article of manufacturecomprising: a container comprising a label and one or more of any of theantibodies or antigen-binding fragments thereof described herein,wherein the label indicates that the composition is to be administeredto a human having, suspected of having, or at risk for developing, acomplement-associated condition. The article of manufacture can furthercomprise one or more additional active therapeutic agents for use intreating a human having, suspected of having, or at risk for developing,a complement-associated condition.

In another aspect, the disclosure features a method for treating apatient afflicted with a complement-associated condition, the methodcomprising administering to the subject one or more of any of theantibodies or antigen-binding fragments thereof described herein in anamount effective to treat the complement-associated condition. Thecomplement-associated condition can be, e.g., one selected from thegroup consisting of rheumatoid arthritis, antiphospholipid antibodysyndrome, lupus nephritis, ischemia-reperfusion injury, atypicalhemolytic uremic syndrome, typical hemolytic uremic syndrome, paroxysmalnocturnal hemoglobinuria, dense deposit disease, neuromyelitis optica,multifocal motor neuropathy, multiple sclerosis, macular degeneration,HELLP syndrome, spontaneous fetal loss, thrombotic thrombocytopenicpurpura, Pauci-immune vasculitis, epidermolysis bullosa, recurrent fetalloss, traumatic brain injury, myocarditis, a cerebrovascular disorder, aperipheral vascular disorder, a renovascular disorder, amesenteric/enteric vascular disorder, vasculitis, Henoch-Schönleinpurpura nephritis, systemic lupus erythematosus-associated vasculitis,vasculitis associated with rheumatoid arthritis, immune complexvasculitis, Takayasu's disease, dilated cardiomyopathy, diabeticangiopathy, Kawasaki's disease, venous gas embolus, restenosis followingstent placement, rotational atherectomy, percutaneous transluminalcoronary angioplasty, myasthenia gravis, cold agglutinin disease,dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipidsyndrome, Graves' disease, atherosclerosis, Alzheimer's disease,systemic inflammatory response sepsis, septic shock, spinal cord injury,glomerulonephritis, transplant rejection (e.g., kidney transplant),Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus,autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura,Goodpasture's syndrome, Degos disease, and catastrophic antiphospholipidsyndrome.

As used herein, the term “antibody” refers to a whole antibodycomprising two light chain polypeptides and two heavy chainpolypeptides. Whole antibodies include different antibody isotypesincluding IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody”includes a polyclonal antibody, a monoclonal antibody, a chimerized orchimeric antibody, a humanized antibody, a primatized antibody, adeimmunized antibody, and a fully human antibody. The antibody can bemade in or derived from any of a variety of species, e.g., mammals suchas humans, non-human primates (e.g., orangutan, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be apurified or a recombinant antibody.

As used herein, the term “antibody fragment,” “antigen-bindingfragment,” or similar terms refer to a fragment of an antibody thatretains the ability to bind to a target antigen (e.g., human C5) andinhibit the activity of the target antigen. Such fragments include,e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fdfragment, an Fab fragment, an Fab′ fragment, or an F(ab′)₂ fragment. AnscFv fragment is a single polypeptide chain that includes both the heavyand light chain variable regions of the antibody from which the scFv isderived. In addition, intrabodies, minibodies, triabodies, and diabodiesare also included in the definition of antibody and are compatible foruse in the methods described herein. See, e.g., Todorovska et al. (2001)J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J ImmunolMethods 231(1):177-189; Poljak (1994) Structure 2(12):1121-1123; Rondonand Marasco (1997) Annual Review of Microbiology 51:257-283, thedisclosures of each of which are incorporated herein by reference intheir entirety.

As used herein, the term “antibody fragment” also includes, e.g., singledomain antibodies such as camelized single domain antibodies. See, e.g.,Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al.(2000) Curr Pharm Biotech 1:253-263; Reichmann et al. (1999) J ImmunolMeth 231:25-38; PCT application publication nos. WO 94/04678 and WO94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporatedherein by reference in their entireties. In some embodiments, thedisclosure provides single domain antibodies comprising two VH domainswith modifications such that single domain antibodies are formed.

In some embodiment, an antigen-binding fragment includes the variableregion of a heavy chain polypeptide and the variable region of a lightchain polypeptide. In some embodiments, an antigen-binding fragmentdescribed herein comprises the CDRs of the light chain and heavy chainpolypeptide of an antibody.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methodsfor treating or preventing a complement-associated condition, will beapparent from the following description, the examples, and from theclaims.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 depicts the amino acid sequence of the heavy chain CDR1 ofeculizumab (as defined under the combined Kabat-Chothia definition).

SEQ ID NO:2 depicts the amino acid sequence of the heavy chain CDR2 ofeculizumab (as defined under the Kabat definition).

SEQ ID NO:3 depicts the amino acid sequence of the heavy chain CDR3 ofeculizumab (as defined under the combined Kabat definition).

SEQ ID NO:4 depicts the amino acid sequence of the light chain CDR1 ofeculizumab (as defined under the Kabat definition).

SEQ ID NO:5 depicts the amino acid sequence of the light chain CDR2 ofeculizumab (as defined under the Kabat definition).

SEQ ID NO:6 depicts the amino acid sequence of the light chain CDR3 ofeculizumab (as defined under the Kabat definition).

SEQ ID NO:7 depicts the amino acid sequence of the heavy chain variableregion of eculizumab.

SEQ ID NO:8 depicts the amino acid sequence of the light chain variableregion of eculizumab and the BNJ441 antibody.

SEQ ID NO:9 depicts the amino acid sequence of the heavy chain constantregion of eculizumab.

SEQ ID NO:10 depicts the amino acid sequence of the entire heavy chainof eculizumab.

SEQ ID NO:11 depicts the amino acid sequence of the entire light chainof eculizumab and the BNJ441 antibody.

SEQ ID NO:12 depicts the amino acid sequence of the heavy chain variableregion of the BNJ441 antibody.

SEQ ID NO:13 depicts the amino acid sequence of the heavy chain constantregion of the BNJ441 antibody.

SEQ ID NO:14 depicts the amino acid sequence of the entire heavy chainof the BNJ441 antibody.

SEQ ID NO:15 depicts the amino acid sequence of an IgG2 heavy chainconstant region variant comprising the YTE substitutions.

SEQ ID NO:16: depicts the amino acid sequence of the entire heavy chainof an eculizumab variant comprising the heavy chain constant regiondepicted in SEQ ID NO:15 (above).

SEQ ID NO:17 depicts the amino acid sequence of the light chain CDR1 ofeculizumab (as defined under the Kabat definition) with a glycine tohistidine substitution at position 8 relative to SEQ ID NO:4.

SEQ ID NO:18 depicts the amino acid sequence of the light chain variableregion of the EHG303 antibody.

SEQ ID NO:19 depicts the amino acid sequence of the heavy chain CDR2 ofeculizumab in which serine at position 8 relative to SEQ ID NO:2 issubstituted with a histidine.

SEQ ID NO:20 depicts the amino acid sequence of the so-called “FLAG”tag.

SEQ ID NO:21 depicts a polyhistidine sequence commonly used as anantigenic tag.

SEQ ID NO:22 depicts the amino acid sequence of the so-calledhemagglutinin tag.

SEQ ID NO:23 depicts the amino acid sequence of the heavy chain CDR1 ofeculizumab in which the tyrosine at position 2 (relative to SEQ ID NO:1)is substituted with histidine.

SEQ IDNO:24 depicts the heavy chain polypeptide amino acid sequence ofthe EHG303 antibody.

SEQ ID NO:25 depicts the light chain polypeptide amino acid sequence ofthe EHG303 antibody.

SEQ ID NO: 26 depicts the amino acid sequence of the heavy chainpolypeptide of the EHL049 antibody.

SEQ ID NO: 27 depicts the amino acid sequence of the light chainpolypeptide of the EHL049 antibody.

SEQ ID NO:28 depicts the EHL000 heavy chain polypeptide amino acidsequence.

SEQ ID NO:29 depicts the amino acid sequence of the light chainpolypeptide of the EHL000 antibody.

SEQ ID NO:30 depicts the light chain polypeptide amino acid sequence ofBHL006.

SEQ ID NO:31 depicts the amino acid sequence of the heavy chainpolypeptide of the BHL006 antibody.

SEQ ID NO:32 depicts the amino acid sequence of the light chainpolypeptide of the BHL009 antibody.

SEQ ID NO:33 depicts the amino acid sequence of the heavy chain of theBHL009 antibody.

SEQ ID NO:34 depicts the amino acid sequence of the light chain of theBHL0011 antibody.

SEQ ID NO:35 depicts the amino acid sequence of the heavy chain of theBHL011 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph depicting the clearance of eculizumab from theserum of human FcRn transgenic mice in the presence or absence ofexogenous human C5. The Y-axis represents the percentage of antibodyremaining in the serum and the X-axis represents the time in days.

FIG. 2 is a line graph depicting the clearance of an eculizumab varianthaving an IgG2 constant region (Ecu-IgG2) and the Ecu-IgG2 antibodycontaining the YTE substitutions (Ecu-IgG2(YTE)) from the serum of mice.The Y-axis represents the percentage of antibody remaining in the serumand the X-axis represents the time in days.

FIG. 3 is a line graph depicting the clearance of an eculizumab varianthaving an IgG2 constant region (Ecu-IgG2) and the Ecu-IgG2 antibodycontaining the YTE substitutions (Ecu-IgG2(YTE)) from the serum of mice.The experiments were performed in the presence or absence of exogenoushuman C5. The Y-axis represents the percentage of antibody remaining inthe serum and the X-axis represents the time in days.

FIG. 4 is a sensorgram plot depicting the kinetics of association (at pH7.4) and dissociation (at pH 7.4 and pH 6.0) for three anti-C5antibodies: EHL000, EHG303, and EHL049. The Y-axis is in arbitraryunits, whereas the X-axis represents time (in seconds).

FIG. 5A is a sensorgram plot depicting the kinetics of dissociation atpH 7.4 and pH 6.0 for the EHG303 (Y27H-S57H double substitution)antibody, the Y27H single substitution variant of eculizumab, andeculizumab (ecu; Ec293F). The Y-axis is in nanometers (nm), whereas theX-axis represents time (in seconds).

FIG. 5B is a sensorgram plot depicting the kinetics of dissociation atpH 7.4 and pH 6.0 for the EHG304 (I34H-L52H double substitution)antibody, the I34H single substitution variant of eculizumab, andeculizumab (ecu; Ec293F). The Y-axis is in nanometers (nm), whereas theX-axis represents time (in seconds). The EHG304 antibody did not meetthe second threshold for selection—namely it exceeded the maximumtolerated variance (from eculizumab) for dissociation at pH7.4.

FIG. 5C is a sensorgram plot depicting the kinetics of dissociation atpH 7.4 and pH 6.0 for the EHG303 (Y27H-S57H double substitution)antibody and eculizumab (ecu; Ec293F). The Y-axis is in nanometers (nm),whereas the X-axis represents time (in seconds).

FIG. 5D is a sensorgram plot depicting the kinetics of dissociation atpH 7.4 and pH 6.0 for the EHL049 [G31H (light chain)/Y27H-S57H doublesubstitution (heavy chain)] antibody, the Y27H-S57H (EHG303) doublesubstitution variant of eculizumab, and eculizumab (ecu). The Y-axis isin nanometers (nm), whereas the X-axis represents time (in seconds).

FIG. 5E is a sensorgram plot depicting the kinetics of dissociation atpH 7.4 and pH 6.0 for the EHL058 [G31H (light chain)/L52H-S57H doublesubstitution (heavy chain)] antibody, the L52H-S57H double substitution(heavy chain) variant of eculizumab, and eculizumab (ecu). The Y-axis isin nanometers (nm), whereas the X-axis represents time (in seconds). TheEHL058 antibody did not meet the second threshold for selection—namelyit exceeded the maximum tolerated variance (from eculizumab) fordissociation at pH7.4.

FIG. 6 is a line graph depicting the clearance of EHL000, BNJ421, andBNJ423 from the serum of NOD/scid/C5-deficient mice. The Y-axisrepresents the percentage of antibody remaining in the serum and theX-axis represents the time in days.

FIG. 7 is a line graph depicting the clearance of EHL000, BNJ421, andBNJ423 from the serum of NOD/scid/C5-deficient mice in the presence orabsence of human C5. The Y-axis represents the percentage of antibodyremaining in the serum and the X-axis represents the time in days.

FIG. 8 is a line graph depicting the activity of the EHL000, BNJ423, andBNJ421 antibodies in an ex vivo hemolytic assay. The Y-axis representsthe percentage of hemolysis and the X-axis represents the time in days.

FIG. 9A is a line graph depicting the pharmacokinetics of the BHL011antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the concentration of antibody in μg/mL.The X-axis represents time in days.

FIG. 9B is a line graph depicting the pharmacokinetics of the BHL011antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the % of the concentration of antibody atday 1 remaining in the serum at each time point. The X-axis representstime in days.

FIG. 10A is a line graph depicting the pharmacokinetics of the BHL006antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the concentration of antibody in μg/mL.The X-axis represents time in days.

FIG. 10B is a line graph depicting the pharmacokinetics of the BHL006antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the % of the concentration of antibody atday 1 remaining in the serum at each time point. The X-axis representstime in days.

FIG. 11A is a line graph depicting the pharmacokinetics of the BHL009antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the concentration of antibody in μg/mL.The X-axis represents time in days.

FIG. 11B is a line graph depicting the pharmacokinetics of the BHL009antibody in hFcRn-transgenic mice. Each line represents a differentanimal. The Y-axis represents the % of the concentration of antibody atday 1 remaining in the serum at each time point. The X-axis representstime in days.

FIG. 12 is a line graph depicting a log plot of the meanpharmacokinetics of the BHL011, BHL006, and BHL009 antibodies inhFcRn-transgenic mice. Each line represents a different antibody asindicated. The Y-axis represents the % of the concentration of antibodyat day 1 remaining in the serum at each time point. The X-axisrepresents time in days.

FIG. 13 is a line graph depicting a linear plot of the meanpharmacokinetics of the BHL011, BHL006, and BHL009 antibodies inhFcRn-transgenic mice. Each line represents a different antibody asindicated. The Y-axis represents the % of the concentration of antibodyat day 1 remaining in the serum at each time point. The X-axisrepresents time in days.

FIG. 14 is a line graph depicting the blocking ability of the BHL011antibody in an ex vivo serum hemolytic assay after a single dose. TheY-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents the time in days.

FIG. 15 is a line graph depicting the blocking ability of the BHL006antibody in an ex vivo serum hemolytic assay after a single dose. TheY-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents the time in days.

FIG. 16 is a line graph depicting the blocking ability of the BHL009antibody in an ex vivo serum hemolytic assay after a single dose. TheY-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents the time in days.

FIG. 17 is a graph depicting the correlation of BHL011 serumconcentration and ex vivo serum hemolytic activity after a single dose.The Y-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents antibody concentration in μg/mL.

FIG. 18 is a graph depicting the correlation of BHL006 serumconcentration and ex vivo serum hemolytic activity after a single dose.The Y-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents antibody concentration in μg/mL.

FIG. 19 is a graph depicting the correlation of BHL009 serumconcentration and ex vivo serum hemolytic activity after a single dose.The Y-axis represents the percentage of hemolysis (relative to pre-doselevels) and the X-axis represents antibody concentration in μg/mL.

FIG. 20 is a line graph depicting the mean ex vivo hemolytic activityafter a single dose of BHL011, BHL009, or BHL006 in hFcRn-transgenicmice. Each line represents a different antibody as indicated. The Y-axisrepresents the percentage of hemolysis (relative to pre-dose levels) andthe X-axis represents time in days.

FIGS. 21A and 21B are a pair of line graphs depicting the semi-log (FIG.21A) and linear (FIG. 21B) plots of the affinity of BNJ441 andeculizumab as a function of pH. The Y axis represents % dissociation andthe X-axis is pH.

FIG. 22 is a line graph depicting the pharmacokinetics of BNJ441 andeculizumab in the NOD/scid mice and in the absence of human C5. TheY-axis represents the concentration of antibody in μg/mL. The X-axisrepresents time in days.

FIG. 23 is a line graph depicting the pharmacokinetics of BNJ441 andeculizumab in the NOD/scid mice and in the presence of human C5. TheY-axis represents the concentration of antibody in μg/mL. The X-axisrepresents time in days.

FIG. 24 is a line graph depicting the percentage of BNJ441 andeculizumab remaining in the serum of NOD/scid mice in the presence ofhuman C5 as a function of time. The Y-axis represents the concentrationof antibody in μg/mL. The X-axis represents time in days.

FIG. 25 is a line graph depicting the ex vivo serum hemolytic blockingactivity of the BNJ441 antibody and eculizumab after a single dose as afunction of time. The Y-axis represents the percentage of hemolysis(relative to pre-dose levels) and the X-axis represents the time indays.

FIG. 26 depicts mean serum BNJ441 concentration-time profiles followingintravenous administration of a 200 mg or 400 mg Dose to HealthyVolunteers (top panel—linear scale; bottom panel—log-linear scale).

FIG. 27 depicts mean chicken red blood cell hemolysis—time profilesfollowing intravenous administration of placebo, 200 mg BNJ441, or 400mg BNJ441 to Healthy Volunteers.

FIG. 28 depicts the relationship between BNJ441 concentration andpercent chicken red blood cell hemolysis following intravenousadministration of BNJ441 to healthy human volunteers.

FIG. 29 depicts the potency of BNJ441 compared to ecculizumab interminal complement activity assays.

FIG. 30 depicts the structure of BNJ441.

FIG. 31 depicts the inter-chain disulfide bonds of BNJ441.

DETAILED DESCRIPTION

The disclosure provides antibodies that are useful for, among otherthings, inhibiting terminal complement (e.g., the assembly and/oractivity of the C5b-9 TCC) and C5a anaphylatoxin-mediated inflammationand, thus, treating complement-associated disorders. The antibodies havea number of improved properties relative to eculizumab, including, e.g.,increased serum half-life in a human. While in no way intended to belimiting, exemplary antibodies, conjugates, pharmaceutical compositionsand formulations, and methods for using any of the foregoing areelaborated on below and are exemplified in the working Examples.

Antibodies

The anti-C5 antibodies described herein bind to complement component C5(e.g., human C5) and inhibit the cleavage of C5 into fragments C5a andC5b. As described above, such antibodies also have, for example,improved pharmacokinetic properties relative to other anti-C5 antibodies(e.g., eculizumab) used for therapeutic purposes.

In some embodiments, an anti-C5 antibody described herein comprises: (i)a heavy chain CDR1 comprising the amino acid sequence depicted in SEQ IDNO:1, (ii) a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:2, (iii) a heavy chain CDR3 comprising the aminoacid sequence depicted in SEQ ID NO:3, (iv) a light chain CDR1comprising the amino acid sequence depicted in SEQ ID NO:4, (v) a lightchain CDR2 comprising the amino acid sequence depicted in SEQ ID NO:5,and (vi) a light chain CDR3 comprising the amino acid sequence depictedin SEQ ID NO:6, in which at least one (e.g., at least two, three, four,five, six, seven, eight, nine, or 10 or more) amino acid(s) of (i)-(vi)is substituted with a different amino acid.

The exact boundaries of CDRs have been defined differently according todifferent methods. In some embodiments, the positions of the CDRs orframework regions within a light or heavy chain variable domain can beas defined by Kabat et al. [(1991) “Sequences of Proteins ofImmunological Interest.” NIH Publication No. 91-3242, U.S. Department ofHealth and Human Services, Bethesda, Md.]. In such cases, the CDRs canbe referred to as “Kabat CDRs” (e.g., “Kabat LCDR2” or “Kabat HCDR1”).In some embodiments, the positions of the CDRs of a light or heavy chainvariable region can be as defined by Chothia et al. (1989) Nature342:877-883. Accordingly, these regions can be referred to as “ChothiaCDRs” (e.g., “Chothia LCDR2” or “Chothia HCDR3”). In some embodiments,the positions of the CDRs of the light and heavy chain variable regionscan be as defined by a Kabat-Chothia combined definition. In suchembodiments, these regions can be referred to as “combined Kabat-ChothiaCDRs”. Thomas et al. [(1996) Mol Immunol 33(17/18):1389-1401]exemplifies the identification of CDR boundaries according to Kabat andChothia definitions.

Any amino acid can be substituted with any other amino acid. In someembodiments, the substitution is a conservative substitution.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine, glutamine, serine andthreonine; lysine, histidine and arginine; and phenylalanine andtyrosine. In some embodiments, one or more amino acids are substitutedwith histidine.

In some embodiments, at least one (e.g., at least two, three, four, orfive) amino acid of heavy chain CDR1 is substituted with a differentamino acid. In some embodiments, at least one (e.g., at least two,three, four, or five) amino acid of heavy chain CDR2 is substituted witha different amino acid. In some embodiments, at least one (e.g., atleast two, three, four, or five) amino acid of heavy chain CDR3 issubstituted with a different amino acid.

In some embodiments, at least one (e.g., at least two, three, four, orfive) amino acid of light chain CDR1 is substituted with a differentamino acid. In some embodiments, at least one (e.g., at least two,three, four, or five) amino acid of light chain CDR2 is substituted witha different amino acid. In some embodiments, at least one (e.g., atleast two, three, four, or five) amino acid of light chain CDR3 issubstituted with a different amino acid.

In some embodiments, a substitution is made at an amino acid positionselected from the group consisting of: glycine at position 1 relative toSEQ ID NO:1, tyrosine at position 2 relative to SEQ ID NO:1, isoleucineat position 3 relative to SEQ ID NO:1, phenylalanine at position 4relative to SEQ ID NO:1, serine at position 5 relative to SEQ ID NO:1,asparagine at position 6 relative to SEQ ID NO:1, tyrosine at position 7relative to SEQ ID NO:1, tryptophan at position 8 relative to SEQ IDNO:1, isoleucine at position 9 relative to SEQ ID NO:1, glutamine atposition 10 relative to SEQ ID NO:1, glutamic acid at position 1relative to SEQ ID NO:2, isoleucine at position 2 relative to SEQ IDNO:2, leucine at position 3 relative to SEQ ID NO:2, proline at position4 relative to SEQ ID NO:2, glycine at position 5 relative to SEQ IDNO:2, serine at position 6 relative to SEQ ID NO:2, glycine at position7 relative to SEQ ID NO:2, serine at position 8 relative to SEQ ID NO:2,threonine at position 9 relative to SEQ ID NO:2, glutamic acid atposition 10 relative to SEQ ID NO:2, tyrosine at position 11 relative toSEQ ID NO:2, threonine at position 12 relative to SEQ ID NO:2, glutamicacid at position 13 relative to SEQ ID NO:2, asparagine at position 14relative to SEQ ID NO:2, phenylalanine at position 15 relative to SEQ IDNO:2, lysine at position 16 relative to SEQ ID NO:2, aspartic acid atposition 17 relative to SEQ ID NO:2, tyrosine at position 1 relative toSEQ ID NO:3, phenylalanine at position 2 relative to SEQ ID NO:3,phenylalanine at position 3 relative to SEQ ID NO:3, glycine at position4 relative to SEQ ID NO:3, serine at position 5 relative to SEQ ID NO:3,serine at position 6 relative to SEQ ID NO:3, proline at position 7relative to SEQ ID NO:3, asparagine at position 8 relative to SEQ IDNO:3, tryptophan at position 9 relative to SEQ ID NO:3, tyrosine atposition 10 relative to SEQ ID NO:3, phenylalanine at position 11relative to SEQ ID NO:3, aspartic acid at position 12 relative to SEQ IDNO:3, and valine at position 13 relative to SEQ ID NO:3.

In some embodiments, the glycine at position 31 relative to SEQ ID NO:8is substituted with a different amino acid. For example, the underlinedglycine in CDR1 of the light chain of eculizumab can be substituted witha different amino acid: GASENIYGALN (SEQ ID NO:4). The substitution canbe a histidine for glycine, i.e., GASENIYHALN (SEQ ID NO:17).

In some embodiments, an anti-C5 antibody described herein comprises anamino acid substitution at an amino acid position selected from thegroup consisting of: glycine at position 26 relative to SEQ ID NO:7,tyrosine at position 27 relative to SEQ ID NO:7, isoleucine at position28 relative to SEQ ID NO:7, phenylalanine at position 29 relative to SEQID NO:7, serine at position 30 relative to SEQ ID NO:7, asparagine atposition 31 relative to SEQ ID NO:7, tyrosine at position 32 relative toSEQ ID NO:7, tryptophan at position 33 relative to SEQ ID NO:7,isoleucine at position 34 relative to SEQ ID NO:7, glutamine at position35 relative to SEQ ID NO:7, glutamic acid at position 50 relative to SEQID NO:7, isoleucine at position 51 relative to SEQ ID NO:7, leucine atposition 52 relative to SEQ ID NO:7, proline at position 53 relative toSEQ ID NO:7, glycine at position 54 relative to SEQ ID NO:7, serine atposition 55 relative to SEQ ID NO:7, glycine at position 56 relative toSEQ ID NO:7, serine at position 57 relative to SEQ ID NO:7, threonine atposition 58 relative to SEQ ID NO:7, glutamic acid at position 59relative to SEQ ID NO:7, tyrosine at position 60 relative to SEQ IDNO:7, threonine at position 61 relative to SEQ ID NO:7, glutamic acid atposition 62 relative to SEQ ID NO:7, asparagine at position 63 relativeto SEQ ID NO:7, phenylalanine at position 64 relative to SEQ ID NO:7,lysine at position 65 relative to SEQ ID NO:7, aspartic acid at position66 relative to SEQ ID NO:7, tyrosine at position 99 relative to SEQ IDNO:7, phenylalanine at position 100 relative to SEQ ID NO:7,phenylalanine at position 101 relative to SEQ ID NO:7, glycine atposition 102 relative to SEQ ID NO:7, serine at position 103 relative toSEQ ID NO:7, serine at position 104 relative to SEQ ID NO:7, proline atposition 105 relative to SEQ ID NO:7, asparagine at position 106relative to SEQ ID NO:7, tryptophan at position 107 relative to SEQ IDNO:7, tyrosine at position 108 relative to SEQ ID NO:7, phenylalanine atposition 109 relative to SEQ ID NO:7, aspartic acid at position 110relative to SEQ ID NO:7, and valine at position 111 relative to SEQ IDNO:7. In some embodiments, the anti-C5 antibody comprises two or more(e.g., at least two, three, four, five, six, seven, eight, nine, or 10or more) of any of the foregoing substitutions and in any combination.

In some embodiments, the anti-C5 antibody comprises at least onesubstitution that meets the following criteria with respect toeculizumab:

-   -   (1) a maximum variation for association kinetics at pH 7.4 of a        33% smaller peak phase shift at 800 seconds as compared to the        averaged peak phase shift at 800 seconds observed for        eculizumab;    -   (2) a maximum variation for dissociation kinetics at pH 7.4 of        no more than 3-fold reduction in peak phase shift over 800        seconds as compared to the averaged peak phase shift at 800        seconds observed for eculizumab; and    -   (3) a minimum variation for dissociation kinetics at pH 6.0 of        at least a 3-fold reduction in the peak phase shift over 800        seconds as compared to the averaged peak phase shift at 800        seconds observed for eculizumab.        For example, with respect to the criterion (1) above, if the        average peak phase shift after 800 seconds of association with        eculizumab is approximately 0.75 nm, a test antibody that has a        phase shift of less than 0.5 nm (e.g., reproduced two or more        times) would not meet the above criteria. By contrast, an        anti-C5 antibody with greater than a 0.5 nm peak phase shift at        800 seconds meets the first criterion. Such substitutions give        rise to anti-C5 antibodies that only deviate from the k_(a) and        k_(d) of eculizumab at pH 7.4 to a minor degree, but deviate        from the k_(d) of eculizumab at pH 6.0 more significantly.

In some embodiments, an anti-C5 antibody described herein comprises atleast one (e.g., at least two, three, or four) amino acid substitutionat an amino acid position selected from the group consisting of: glycineat position 31 relative to SEQ ID NO:8, leucine at position 33 relativeto SEQ ID NO:8, valine at position 91 relative to SEQ ID NO:8, andthreonine at position 94 relative to SEQ ID NO:8. In some embodiments,an anti-C5 antibody described herein comprises at least one (e.g., two,three, four or five) amino acid substitution(s) at an amino acidposition selected from the group consisting of: tyrosine at position 27relative to SEQ ID NO:7, isoleucine at position 34 relative to SEQ IDNO:7, leucine at position 52 relative to SEQ ID NO:7, and serine atposition 57 relative to SEQ ID NO:7.

In some embodiments, an anti-C5 antibody described herein contains inits light chain variable region at least one substitution selected fromthe following: glycine at position 31 relative to SEQ ID NO:8, leucineat position 33 relative to SEQ ID NO:8, valine at position 91 relativeto SEQ ID NO:8, and threonine at position 94 relative to SEQ ID NO:8.See Table 1 below. In some embodiments, an anti-C5 antibody describedherein contains in its heavy chain variable region at least onesubstitution selected from the following: tyrosine at position 27relative to SEQ ID NO:7, isoleucine at position 34 relative to SEQ IDNO:7, leucine at position 52 relative to SEQ ID NO:7, and serine atposition 57 relative to SEQ ID NO:7. See Table 1 below.

In some embodiments, an antibody comprises at least two (e.g., at leastthree, four, five, six, seven, eight, nine, or 10) amino acidsubstitutions relative to the CDR set defined by SEQ ID NOs:1-6. Thus,in some embodiments, an anti-C5 antibody described herein comprises twoor more substitutions in the combinations and at the amino acidpositions set forth in Table 1.

TABLE 1 Amino Acid Substitution Combinations Substitutions within theLight Substitutions within the Heavy Chain Variable Region CDRs of ChainVariable Region CDRs of Eculizumab (relative to SEQ ID Eculizumab(relative to SEQ ID Ab NO: 8). NO: 7). Cmb Amino Acid Position No.: G31L33 V91 T94 Y27 I34 L52 S57 1 • • 2 • • 3 • • 4 • • 5 • • 6 • • 7 • • 8• • 9 • • 10 • • 11 • • 12 • • 13 • • • • 14 • • • • 15 • • • • 16 • • •• 17 • • • • 18 • • • • 19 • • • • 20 • • • • 21 • • • • 22 • • • • 23 •• • • 24 • • • • 25 • • • • 26 • • • • 27 • • • • 28 • • • • 29 • • • •30 • • • • 31 • • • 32 • • • 33 • • • 34 • • • 35 • • • 36 • • • 37 • •• 38 • • • 39 • • • 40 • • • 41 • • • 42 • • • 43 • • • 44 • • • 45 • •• 46 • • • 47 • • • 48 • • • 49 • • • 50 • • • 51 • • • 52 • • • 53 • •• 54 • • • 55 • • • 56 • • • 57 • • • 58 • • • 59 • • • 60 • • • 61 • •• • 62 • • • • 63 • • • • 64 • • • • 65 • • • • • 66 • • • • • 67 • • •• • 68 • • • • 69 • • • • 70 • • • • 71 • • • • 72 • • • • 73 • • • • 74• • • • 75 • • • • 76 • • • • • • • “•” indicates which of the aminoacids are substituted in a given antibody. For example, Ab Cmb. No. 76defines an antibody comprising the six CDRs of eculizumab, in which thelight chain CDRs comprise substitutions at positions 31, 33, and 91,relative to SEQ ID NO: 8 and the heavy chain CDRs comprise substitutionsat positions 27, 34, 52, and 57, relative to SEQ ID NO: 7. “Ab Comb.No.” refers to a numerical designation given to a particular variantanti-C5 antibody referred to in the table. To be clear, the variantanti-C5 antibodies referred to in Table 1 need only have the amino acidsequences of the six (6) CDRs of eculizumab in which the given,indicated amino acid substitutions are made. The variant antibodies may,optionally, include the framework regions of SEQ ID NO: 7 or SEQ ID NO:8.The substitutions described in Table 1 can be for any amino acid that isdifferent from the indicated amino acid residue. In some embodiments,the different amino acid is a histidine.

In some embodiments, an anti-C5 antibody described herein comprises asubstitution made at an amino acid position selected from the groupconsisting of: tyrosine at position 27 relative to SEQ ID NO:7,isoleucine at position 34 relative to SEQ ID NO:7, leucine at position52 relative to SEQ ID NO:7, and serine at position 57 relative to SEQ IDNO:7. In some embodiments, both tyrosine at position 27 relative to SEQID NO:7 and leucine at position 52 relative to SEQ ID NO:7 are eachsubstituted with a different amino acid. In some embodiments, bothisoleucine at position 34 relative to SEQ ID NO:7 and serine at position57 relative to SEQ ID NO:7 are each substituted with a different aminoacid. In some embodiments, both isoleucine at position 34 relative toSEQ ID NO:7 and leucine at position 52 relative to SEQ ID NO:7 are eachsubstituted with a different amino acid. In some embodiments, bothtyrosine at position 27 relative to SEQ ID NO:7 and serine at position57 relative to SEQ ID NO:7 are each substituted with a different aminoacid. In some embodiments of any of the anti-C5 antibodies describedherein, the different amino acid is a histidine. For example, tyrosineat position 27 and serine at position 57 can each be substituted withhistidine.

In some embodiments, an anti-C5 antibody described herein comprises aheavy chain CDR1 comprising, or consisting of, the following amino acidsequence: GHIFSNYWIQ (SEQ ID NO:23). In some embodiments, an anti-C5antibody described herein comprises a heavy chain CDR2 comprising, orconsisting of, the following amino acid sequence: EILPGSGHTEYTENFKD (SEQID NO:19). In some embodiments, an anti-C5 antibody described hereincomprises a heavy chain variable region comprising the following aminoacid sequence:

(SEQ ID NO: 12) QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWMGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.

In some embodiments, an anti-C5 antibody described herein comprises alight chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

An anti-C5 antibody described herein can bind to C5 at pH 7.4 and 25° C.(and, otherwise, under physiologic conditions) with an affinitydissociation constant (K_(D)) that is at least 0.1 (e.g., at least 0.15,0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45,0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725,0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM. Insome embodiments, the K_(D) of the anti-C5 antibody is no greater than 1(e.g., no greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2) nM.

In some embodiments of any anti-C5 antibody described herein, the[(K_(D) of the antibody for C5 at pH 6.0 at C)/(K_(D) of the antibodyfor C5 at pH 7.4 at 25° C.)] is greater than 21 (e.g., greater than 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600,700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, or 8000).

Methods for determining whether an antibody binds to a protein antigenand/or the affinity for an antibody to a protein antigen are known inthe art. For example, the binding of an antibody to a protein antigencan be detected and/or quantified using a variety of techniques such as,but not limited to, Western blot, dot blot, surface plasmon resonance(SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala,Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assay(ELISA). See, e.g., Harlow and Lane (1988) “Antibodies: A LaboratoryManual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols,”Humana Press (ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering,A Practical Guide,” W.H. Freeman and Co., NY; Borrebaek (1995) “AntibodyEngineering,” 2^(nd) Edition, Oxford University Press, NY, Oxford; Johneet al. (1993) J Immunol Meth 160:191-198; Jonsson et al. (1993) Ann BiolClin 51:19-26; and Jonsson et al. (1991) Biotechniques 11:620-627. Inaddition, methods for measuring the affinity (e.g., dissociation andassociation constants) are set forth in the working examples.

As used herein, the term “k_(a)” refers to the rate constant forassociation of an antibody to an antigen. The term “k_(d)” refers to therate constant for dissociation of an antibody from the antibody/antigencomplex. And the term “K_(D)” refers to the equilibrium dissociationconstant of an antibody-antigen interaction. The equilibriumdissociation constant is deduced from the ratio of the kinetic rateconstants, K_(D)=k_(a)/k_(d). Such determinations preferably aremeasured at 25° C. or 37° C. (see the working examples). For example,the kinetics of antibody binding to human C5 can be determined at pH8.0, 7.4, 7.0, 6.5 and 6.0 via surface plasmon resonance (SPR) on aBIAcore 3000 instrument using an anti-Fc capture method to immobilizethe antibody.

The anti-C5 antibody described herein can have activity in blocking thegeneration or activity of the C5a and/or C5b active fragments of a C5protein (e.g., a human C5 protein). Through this blocking effect, theantibodies inhibit, e.g., the proinflammatory effects of C5a and thegeneration of the C5b-9 membrane attack complex (MAC) at the surface ofa cell.

Methods for determining whether a particular antibody described hereininhibits C5 cleavage are known in the art. Inhibition of humancomplement component C5 can reduce the cell-lysing ability of complementin a subject's body fluids. Such reductions of the cell-lysing abilityof complement present in the body fluid(s) can be measured by methodswell known in the art such as, for example, by a conventional hemolyticassay such as the hemolysis assay described by Kabat and Mayer (eds.),“Experimental Immunochemistry, 2^(nd) Edition,” 135-240, Springfield,Ill., CC Thomas (1961), pages 135-139, or a conventional variation ofthat assay such as the chicken erythrocyte hemolysis method as describedin, e.g., Hillmen et al. (2004) N Engl J Med 350(6):552. Methods fordetermining whether a candidate compound inhibits the cleavage of humanC5 into forms C5a and C5b are known in the art and described in, e.g.,Moongkarndi et al. (1982) Immunobiol 162:397; Moongkarndi et al. (1983)Immunobiol 165:323; Isenman et al. (1980) J Immunol 124(1):326-31;Thomas et al. (1996) Mol Immunol 33(17-18):1389-401; and Evans et al.(1995) Mol Immunol 32(16):1183-95. For example, the concentration and/orphysiologic activity of C5a and C5b in a body fluid can be measured bymethods well known in the art. Methods for measuring C5a concentrationor activity include, e.g., chemotaxis assays, RIAs, or ELISAs (see,e.g., Ward and Zvaifler (1971) J Clin Invest 50(3):606-16 and Wurzner etal. (1991) Complement Inflamm 8:328-340). For C5b, hemolytic assays orassays for soluble C5b-9 as discussed herein can be used. Other assaysknown in the art can also be used. Using assays of these or othersuitable types, candidate agents capable of inhibiting human complementcomponent C5 can be screened.

Immunological techniques such as, but not limited to, ELISA can be usedto measure the protein concentration of C5 and/or its split products todetermine the ability of an anti-C5 antibody to inhibit conversion of C5into biologically active products. In some embodiments, C5a generationis measured. In some embodiments, C5b-9 neoepitope-specific antibodiesare used to detect the formation of terminal complement.

Hemolytic assays can be used to determine the inhibitory activity of ananti-C5 antibody on complement activation. In order to determine theeffect of an anti-C5 antibody on classical complement pathway-mediatedhemolysis in a serum test solution in vitro, for example, sheeperythrocytes coated with hemolysin or chicken erythrocytes sensitizedwith anti-chicken erythrocyte antibody are used as target cells. Thepercentage of lysis is normalized by considering 100% lysis equal to thelysis occurring in the absence of the inhibitor. In some embodiments,the classical complement pathway is activated by a human IgM antibody,for example, as utilized in the Wieslab® Classical Pathway ComplementKit (Wieslab® COWL CP310, Euro-Diagnostica, Sweden). Briefly, the testserum is incubated with an anti-C5 antibody in the presence of a humanIgM antibody. The amount of C5b-9 that is generated is measured bycontacting the mixture with an enzyme conjugated anti-C5b-9 antibody anda fluorogenic substrate and measuring the absorbance at the appropriatewavelength. As a control, the test serum is incubated in the absence ofthe anti-C5 antibody. In some embodiments, the test serum is aC5-deficient serum reconstituted with a C5 polypeptide.

To determine the effect of anti-C5 antibody on alternativepathway-mediated hemolysis, unsensitized rabbit or guinea pigerythrocytes are used as the target cells. In some embodiments, theserum test solution is a C5-deficient serum reconstituted with a C5polypeptide. The percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor. Insome embodiments, the alternative complement pathway is activated bylipopolysaccharide molecules, for example, as utilized in the Wieslab®Alternative Pathway Complement Kit (Wieslab® COMPL AP330,Euro-Diagnostica, Sweden). Briefly, the test serum is incubated with ananti-C5 antibody in the presence of lipopolysaccharide. The amount ofC5b-9 that is generated is measured by contacting the mixture with anenzyme conjugated anti-C5b-9 antibody and a fluorogenic substrate andmeasuring the fluorescence at the appropriate wavelength. As a control,the test serum is incubated in the absence of the anti-C5 antibody.

In some embodiments, C5 activity, or inhibition thereof, is quantifiedusing a CH50eq assay. The CH50eq assay is a method for measuring thetotal classical complement activity in serum. This test is a lyticassay, which uses antibody-sensitized erythrocytes as the activator ofthe classical complement pathway and various dilutions of the test serumto determine the amount required to give 50% lysis (CH50). The percenthemolysis can be determined, for example, using a spectrophotometer. TheCH50eq assay provides an indirect measure of terminal complement complex(TCC) formation, since the TCC themselves are directly responsible forthe hemolysis that is measured.

The assay is well known and commonly practiced by those of skill in theart. Briefly, to activate the classical complement pathway, undilutedserum samples (e.g., reconstituted human serum samples) are added tomicroassay wells containing the antibody-sensitized erythrocytes tothereby generate TCC. Next, the activated sera are diluted in microassaywells, which are coated with a capture reagent (e.g., an antibody thatbinds to one or more components of the TCC). The TCC present in theactivated samples bind to the monoclonal antibodies coating the surfaceof the microassay wells. The wells are washed and to each well is addeda detection reagent that is detectably labeled and recognizes the boundTCC. The detectable label can be, e.g., a fluorescent label or anenzymatic label. The assay results are expressed in CH50 unitequivalents per milliliter (CH50 U Eq/mL).

Inhibition, e.g., as it pertains to terminal complement activity,includes at least a 5 (e.g., at least a 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, or 60) % decrease in the activity of terminalcomplement in, e.g., a hemolytic assay or CH50eq assay as compared tothe effect of a control antibody (or antigen-binding fragment thereof)under similar conditions and at an equimolar concentration. Substantialinhibition, as used herein, refers to inhibition of a given activity(e.g., terminal complement activity) of at least 40 (e.g., at least 45,50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 or greater) %. In someembodiments, an anti-C5 antibody described herein contains one or moreamino acid substitutions relative to the CDRs of eculizumab (i.e., SEQID NOs:1-6), yet retains at least 30 (e.g., at least 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, or 95) % of the complement inhibitory activity ofeculizumab in a hemolytic assay or CH50eq assay.

An anti-C5 antibody described herein has a serum half-life in humansthat is at least 20 (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, or 36) days. Methods for measuring the serumhalf-life of an antibody are known in the art and exemplified in theworking examples. See, e.g., Dall'Acqua et al. (2006) J Biol Chem 281:23514-23524; Hinton et al. (2004) J Biol Chem 279:6213-6216; Hinton etal. (2006) J Immunol 176:346-356; and Petkova et al. (2006) Int Immunol18(12):1759-69, the disclosures of each of which are incorporated hereinby reference in their entirety. In some embodiments, an anti-C5 antibodydescribed herein has a serum half-life that is at least 20 (e.g., atleast 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200, 250, 300, 400, 500) % greater than the serum half-life ofeculizumab, e.g., as measured in one of the mouse model systemsdescribed in the working examples (e.g., the C5-deficient/NOD/scid mouseor hFcRn transgenic mouse model system).

Modifications to the Fc Region

An anti-C5 antibody described herein can, in some embodiments, comprisea variant human Fc constant region that binds to human neonatal Fcreceptor (FcRn) with greater affinity than that of the native human Fcconstant region from which the variant human Fc constant region wasderived. For example, the Fc constant region can comprise one or more(e.g., two, three, four, five, six, seven, or eight or more) amino acidsubstitutions relative to the native human Fc constant region from whichthe variant human Fc constant region was derived. The substitutions canincrease the binding affinity of an IgG antibody containing the variantFc constant region to FcRn at pH 6.0, while maintaining the pHdependence of the interaction. See, e.g., Hinton et al. (2004) J BiolChem 279:6213-6216 and Datta-Mannan et al. (2007) Drug Metab Dispos35:1-9. Methods for testing whether one or more substitutions in the Fcconstant region of an antibody increase the affinity of the Fc constantregion for FcRn at pH 6.0 (while maintaining pH dependence of theinteraction) are known in the art and exemplified in the workingexamples. See, e.g., Datta-Mannan et al. (2007) J Biol Chem282(3):1709-1717; International Publication No. WO 98/23289;International Publication No. WO 97/34631; and U.S. Pat. No. 6,277,375,the disclosures of each of which are incorporated herein by reference intheir entirety.

Substitutions that enhance the binding affinity of an antibody Fcconstant region for FcRn are known in the art and include, e.g., (1) theM252Y/S254T/T256E triple substitution described by Dall'Acqua et al.(2006) J Biol Chem 281: 23514-23524; (2) the M428L or T250Q/M428Lsubstitutions described in Hinton et al. (2004) J Biol Chem279:6213-6216 and Hinton et al. (2006) J Immunol 176:346-356; and (3)the N434A or T307/E380A/N434A substitutions described in Petkova et al.(2006) Int Immunol 18(12):1759-69. The additional substitution pairings:P257I/Q3111, P257I/N434H, and D376V/N434H are described in, e.g.,Datta-Mannan et al. (2007) J Biol Chem 282(3):1709-1717, the disclosureof which is incorporated herein by reference in its entirety.

In some embodiments, the variant constant region has a substitution atEU amino acid residue 255 for valine. In some embodiments, the variantconstant region has a substitution at EU amino acid residue 309 forasparagine. In some embodiments, the variant constant region has asubstitution at EU amino acid residue 312 for isoleucine. In someembodiments, the variant constant region has a substitution at EU aminoacid residue 386.

In some embodiments, the variant Fc constant region comprises no morethan 30 (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four,three, or two) amino acid substitutions, insertions, or deletionsrelative to the native constant region from which it was derived. Insome embodiments, the variant Fc constant region comprises one or moreamino acid substitutions selected from the group consisting of: M252Y,S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In someembodiments, the variant human Fc constant region comprises a methionineat position 428 and an asparagine at position 434, each in EU numbering.In some embodiments, the variant Fc constant region comprises a428L/434S double substitution as described in, e.g., U.S. Pat. No.8,088,376.

In some embodiments, the variant constant region comprises asubstitution at amino acid position 237, 238, 239, 248, 250, 252, 254,255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308,309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384,385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) relativeto the native human Fc constant region. In some embodiments, thesubstitution is selected from the group consisting of: methionine forglycine at position 237; alanine for proline at position 238; lysine forserine at position 239; isoleucine for lysine at position 248; alanine,phenylalanine, isoleucine, methionine, glutamine, serine, valine,tryptophan, or tyrosine for threonine at position 250; phenylalanine,tryptophan, or tyrosine for methionine at position 252; threonine forserine at position 254; glutamic acid for arginine at position 255;aspartic acid, glutamic acid, or glutamine for threonine at position256; alanine, glycine, isoleucine, leucine, methionine, asparagine,serine, threonine, or valine for proline at position 257; histidine forglutamic acid at position 258; alanine for aspartic acid at position265; phenylalanine for aspartic acid at position 270; alanine, orglutamic acid for asparagine at position 286; histidine for threonine atposition 289; alanine for asparagine at position 297; glycine for serineat position 298; alanine for valine at position 303; alanine for valineat position 305; alanine, aspartic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, valine, tryptophan, or tyrosine forthreonine at position 307; alanine, phenylalanine, isoleucine, leucine,methionine, proline, glutamine, or threonine for valine at position 308;alanine, aspartic acid, glutamic acid, proline, or arginine for leucineor valine at position 309; alanine, histidine, or isoleucine forglutamine at position 311; alanine or histidine for aspartic acid atposition 312; lysine or arginine for leucine at position 314; alanine orhistidine for asparagine at position 315; alanine for lysine at position317; glycine for asparagine at position 325; valine for isoleucine atposition 332; leucine for lysine at position 334; histidine for lysineat position 360; alanine for aspartic acid at position 376; alanine forglutamic acid at position 380; alanine for glutamic acid at position382; alanine for asparagine or serine at position 384; aspartic acid orhistidine for glycine at position 385; proline for glutamine at position386; glutamic acid for proline at position 387; alanine or serine forasparagine at position 389; alanine for serine at position 424; alanine,aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, asparagine, proline, glutamine, serine, threonine, valine,tryptophan, or tyrosine for methionine at position 428; lysine forhistidine at position 433; alanine, phenylalanine, histidine, serine,tryptophan, or tyrosine for asparagine at position 434; and histidinefor tyrosine or phenylalanine at position 436, all in EU numbering.

An anti-C5 antibody described herein can, in some embodiments, comprisea heavy chain polypeptide comprising the amino acid sequence depicted inSEQ ID NO:12 or 14 and/or a light chain polypeptide comprising the aminoacid sequence depicted in SEQ ID NO:8 or 11.

Methods for Producing the Anti-C5 Antibodies and Antigen-BindingFragments Thereof

The disclosure also features methods for producing any of the anti-C5antibodies or antigen-binding fragments thereof described herein. Insome embodiments, methods for preparing an antibody described herein caninclude immunizing a subject (e.g., a non-human mammal) with anappropriate immunogen. Suitable immunogens for generating any of theantibodies described herein are set forth herein. For example, togenerate an antibody that binds to C5, a skilled artisan can immunize asuitable subject (e.g., a non-human mammal such as a rat, a mouse, agerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-humanprimate) with a full-length C5 polypeptide such as a full-length humanC5 polypeptide. In some embodiments, the non-human mammal is C5deficient, e.g., a C5-deficient mouse described in, e.g., Levy and Ladda(1971) Nat New Biol 229(2):51-52; Crocker et al. (1974) J Clin Pathol27(2):122-124; Wetsel et al. (1990) J Biol Chem 265:2435-2440; and Jungiand Pepys (1981) Immunology 43 (2):271-279.

A suitable subject (e.g., a non-human mammal) can be immunized with theappropriate antigen along with subsequent booster immunizations a numberof times sufficient to elicit the production of an antibody by themammal. The immunogen can be administered to a subject (e.g., anon-human mammal) with an adjuvant. Adjuvants useful in producing anantibody in a subject include, but are not limited to, proteinadjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvum or Salmonella minnesota) and bacterial componentsincluding cell wall skeleton, trehalose dimycolate, monophosphoryl lipidA, methanol extractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g.,aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Otheradjuvants that can be used in the methods for inducing an immuneresponse include, e.g., cholera toxin and parapoxvirus proteins. Seealso Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g.,Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J MedChem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Guptaet al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, the methods include preparing a hybridoma cell linethat secretes a monoclonal antibody that binds to the immunogen. Forexample, a suitable mammal such as a laboratory mouse is immunized witha C5 polypeptide as described above. Antibody-producing cells (e.g., Bcells of the spleen) of the immunized mammal can be isolated two to fourdays after at least one booster immunization of the immunogen and thengrown briefly in culture before fusion with cells of a suitable myelomacell line. The cells can be fused in the presence of a fusion promotersuch as, e.g., vaccinia virus or polyethylene glycol. The hybrid cellsobtained in the fusion are cloned, and cell clones secreting the desiredantibodies are selected. For example, spleen cells of Balb/c miceimmunized with a suitable immunogen can be fused with cells of themyeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. After thefusion, the cells are expanded in suitable culture medium, which issupplemented with a selection medium, for example HAT medium, at regularintervals in order to prevent normal myeloma cells from overgrowing thedesired hybridoma cells. The obtained hybrid cells are then screened forsecretion of the desired antibodies, e.g., an antibody that binds to C5and inhibits cleavage of C5 into fragments C5a and C5b.

In some embodiments, any of the antibodies or antigen-binding fragmentsthereof described herein can be manufactured in a CHO cell. In someembodiments, the antibodies or antigen-binding fragments thereof do notcontain detectable sialic acid residues.

In some embodiments, a skilled artisan can identify an anti-C5 antibodyfrom a non-immune biased library as described in, e.g., U.S. Pat. No.6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al.(2005) Nucleic Acids Res 33(9):e81.

A subpopulation of antibodies screened using the above methods can becharacterized for their specificity and binding affinity for aparticular immunogen (e.g., C5) using any immunological or biochemicalbased method known in the art. For example, specific binding of anantibody to native, full-length C5, as compared to C5a, may bedetermined for example using immunological or biochemical based methodssuch as, but not limited to, an ELISA assay, SPR assays,immunoprecipitation assay, affinity chromatography, and equilibriumdialysis as described above. Immunoassays which can be used to analyzeimmunospecific binding and cross-reactivity of the antibodies include,but are not limited to, competitive and non-competitive assay systemsusing techniques such as Western blots, RIA, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays. Such assays are routine andwell known in the art.

Antibodies can also be assayed using any SPR-based assays known in theart for characterizing the kinetic parameters of the interaction of theantibody with C5. Any SPR instrument commercially available including,but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden);1Asys instruments (Affinity Sensors; Franklin, Mass.); IBIS system(Windsor Scientific Limited; Berks, UK), SPR-CELLIA systems (NipponLaser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta(Texas Instruments; Dallas, Tex.) can be used in the methods describedherein. See, e.g., Mullett et al. (2000) Methods 22: 77-91; Dong et al.(2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) CurrOpin Biotechnol 9: 97-101; and Rich et al. (2000) Curr Opin Biotechnol11: 54-61.

It is understood that the above methods can also be used to determineif, e.g., an anti-C5 antibody does not bind to full-length, native C3and/or C4 proteins.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any desiredfragments, and expressed in any desired host, including mammalian cells,insect cells, plant cells, yeast, and bacteria, e.g., as described indetail below. For example, techniques to recombinantly produce Fab, Fab′and F(ab′)2 fragments can also be employed using methods known in theart such as those disclosed in PCT publication no. WO 92/22324; Mullinaxet al. (1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) Am JRepr Immunol 34:26-34; and Better et al. (1988) Science 240:1041-1043.Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu etal. (1993) Proc Nat Acad Sci USA 90:7995-7999; and Skerra et al. (1988)Science 240:1038-1040.

In some embodiments, epitope mapping can be used to identify, e.g., theregion of C5 that interacts with an antibody. Methods for identifyingthe epitope to which a particular antibody binds are also known in theart and are described above.

The antibodies and fragments thereof identified herein can be or can bemade “chimeric.” Chimeric antibodies and antigen-binding fragmentsthereof comprise portions from two or more different species (e.g.,mouse and human). Chimeric antibodies can be produced with mousevariable regions of desired specificity fused to human constant domains(for example, U.S. Pat. No. 4,816,567). In this manner, non-humanantibodies can be modified to make them more suitable for human clinicalapplication (e.g., methods for treating or preventing acomplement-mediated disorder in a subject).

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (e.g., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (see, e.g., Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536), by substituting rodent frameworksor CDR sequences for the corresponding sequences of a human antibody.Also see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In someembodiments, humanized forms of non-human (e.g., mouse) antibodies arehuman antibodies (recipient antibody) in which the CDR region amino acidresidues of the non-human antibody (e.g., mouse, rat, rabbit, ornon-human primate antibody) having the desired specificity, affinity,and binding capacity are grafted onto the framework scaffold of a humanantibody.

In some instances, one or more framework region amino acid residues ofthe human immunoglobulin are also replaced by corresponding amino acidresidues of the non-human antibody (so called “back mutations”). Inaddition, phage display libraries can be used to vary amino acids atchosen positions within the antibody sequence. The properties of ahumanized antibody are also affected by the choice of the humanframework. Furthermore, humanized and chimerized antibodies can bemodified to comprise residues that are not found in the recipientantibody or in the donor antibody in order to further improve antibodyproperties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human immunoglobulin sequences,preferably human germline sequences. Human antibodies can include aminoacid residues not encoded by human germline immunoglobulin sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo). However, the term “humanantibody” does not include antibodies in which CDR sequences derivedfrom another mammalian species, such as a mouse, have been grafted ontohuman framework sequences (i.e., humanized antibodies). Fully human orhuman antibodies may be derived from transgenic mice carrying humanantibody genes (carrying the variable (V), diversity (D), joining (J),and constant (C) exons) or from human cells.

The human sequences may code for both the heavy and light chains ofhuman antibodies and would function correctly in the mice, undergoingrearrangement to provide a wide antibody repertoire similar to that inhumans. The transgenic mice can be immunized with the target proteinimmunogen to create a diverse array of specific antibodies and theirencoding RNA. Nucleic acids encoding the antibody chain components ofsuch antibodies may then be cloned from the animal into a displayvector. Typically, separate populations of nucleic acids encoding heavyand light chain sequences are cloned, and the separate populations thenrecombined on insertion into the vector, such that any given copy of thevector receives a random combination of a heavy and a light chain. Thevector is designed to express antibody chains so that they can beassembled and displayed on the outer surface of a display packagecontaining the vector. For example, antibody chains can be expressed asfusion proteins with a phage coat protein from the outer surface of thephage. Thereafter, display packages can be selected and screened fordisplay of antibodies binding to a target.

In some embodiments, the anti-C5 antibodies described herein comprise analtered heavy chain constant region that has reduced (or no) effectorfunction relative to its corresponding unaltered constant region.Effector functions involving the constant region of the anti-C5 antibodymay be modulated by altering properties of the constant or Fc region.Altered effector functions include, for example, a modulation in one ormore of the following activities: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis,binding to one or more Fc-receptors, and pro-inflammatory responses.Modulation refers to an increase, decrease, or elimination of aneffector function activity exhibited by a subject antibody containing analtered constant region as compared to the activity of the unalteredform of the constant region. In particular embodiments, modulationincludes situations in which an activity is abolished or completelyabsent.

An altered constant region with altered FcR binding affinity and/or ADCCactivity and/or altered CDC activity is a polypeptide which has eitheran enhanced or diminished FcR binding activity and/or ADCC activityand/or CDC activity compared to the unaltered form of the constantregion. An altered constant region which displays increased binding toan FcR binds at least one FcR with greater affinity than the unalteredpolypeptide. An altered constant region which displays decreased bindingto an FcR binds at least one FcR with lower affinity than the unalteredform of the constant region. Such variants which display decreasedbinding to an FcR may possess little or no appreciable binding to anFcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1%) of the binding to the FcR as compared to the level ofbinding of a native sequence immunoglobulin constant or Fc region to theFcR. Similarly, an altered constant region that displays modulated ADCCand/or CDC activity may exhibit either increased or reduced ADCC and/orCDC activity compared to the unaltered constant region. For example, insome embodiments, the anti-C5 antibody comprising an altered constantregion can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activityof the unaltered form of the constant region. An anti-C5 antibodydescribed herein comprising an altered constant region displayingreduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDCactivity.

In certain embodiments, the altered constant region has at least oneamino acid substitution, insertion, and/or deletion, compared to anative sequence constant region or to the unaltered constant region,e.g. from about one to about one hundred amino acid substitutions,insertions, and/or deletions in a native sequence constant region or inthe constant region of the parent polypeptide. In some embodiments, thealtered constant region herein will possess at least about 70% homology(similarity) or identity with the unaltered constant region and in someinstances at least about 75% and in other instances at least about 80%homology or identity therewith, and in other embodiments at least about85%, 90% or 95% homology or identity therewith. The altered constantregion may also contain one or more amino acid deletions or insertions.Additionally, the altered constant region may contain one or more aminoacid substitutions, deletions, or insertions that results in alteredpost-translational modifications, including, for example, an alteredglycosylation pattern (e.g., the addition of one or more sugarcomponents, the loss of one or more sugar components, or a change incomposition of one or more sugar components relative to the unalteredconstant region).

Antibodies with altered or no effector functions may be generated byengineering or producing antibodies with variant constant, Fc, or heavychain regions; recombinant DNA technology and/or cell culture andexpression conditions may be used to produce antibodies with alteredfunction and/or activity. For example, recombinant DNA technology may beused to engineer one or more amino acid substitutions, deletions, orinsertions in regions (such as, for example, Fc or constant regions)that affect antibody function including effector functions.Alternatively, changes in post-translational modifications, such as,e.g., glycosylation patterns, may be achieved by manipulating the cellculture and expression conditions by which the antibody is produced.Suitable methods for introducing one or more substitutions, additions,or deletions into an Fc region of an antibody are well known in the artand include, e.g., standard DNA mutagenesis techniques as described in,e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,2^(nd) Edition,” Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane (1988), supra; Borrebaek (1992), supra;Johne et al. (1993), supra; PCT publication no. WO 06/53301; and U.S.Pat. No. 7,704,497.

In some embodiments, an anti-C5 antibody described herein exhibitsreduced or no effector function. In some embodiments, an anti-C5antibody comprises a hybrid constant region, or a portion thereof, suchas a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) AdvImmun 51:1-18; Canfield et al. (1991) J Exp Med 173:1483-1491; andMueller et al. (1997) Mol Immunol 34(6):441-452). See above.

In addition to using a G2/G4 construct as described above, an anti-C5antibody described herein having reduced effector function may beproduced by introducing other types of changes in the amino acidsequence of certain regions of the antibody. Such amino acid sequencechanges include but are not limited to the Ala-Ala mutation describedin, e.g., PCT Publication nos. WO 94/28027 and WO 98/47531; and Xu etal. (2000) Cell Immunol 200:16-26. Thus, in some embodiments, an anti-C5antibody with one or more mutations within the constant region includingthe Ala-Ala mutation has reduced or no effector function. According tothese embodiments, the constant region of the antibody can comprise asubstitution to an alanine at position 234 or a mutation to an alanineat position 235. Additionally, the altered constant region may contain adouble mutation: a mutation to an alanine at position 234 and a secondmutation to an alanine at position 235. In one embodiment, an anti-C5antibody comprises an IgG4 framework, wherein the Ala-Ala mutation woulddescribe a mutation(s) from phenylalanine to alanine at position 234and/or a mutation from leucine to alanine at position 235. In anotherembodiment, the anti-C5 antibody comprises an IgG1 framework, whereinthe Ala-Ala mutation would describe a mutation(s) from leucine toalanine at position 234 and/or a mutation from leucine to alanine atposition 235. An anti-C5 antibody may alternatively or additionallycarry other mutations, including the point mutation K322A in the CH2domain (Hezareh et al. (2001) J Virol 75:12161-12168). An antibody withsaid mutation(s) in the constant region may furthermore be a blocking ornon-blocking antibody.

Additional substitutions that, when introduced into a heavy chainconstant region, result in decreased effector function are set forth in,e.g., Shields et al. (2001) J Biol Chem 276(9):6591-6604. Seeparticularly Table 1 (“Binding of human IgG1 variants to human FcRn andFcγR) of Shields et al., the disclosure of which is incorporated hereinby reference in its entirety. By screening a library of anti-IgEantibodies, each antibody of the library differing by one or moresubstitutions in the heavy chain constant region, for binding to a panelof Fc receptors (including FcRn, FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA),the authors identified a number of substitutions that modulate specificFc-Fc receptor interactions. For example, a variant IgG2a heavy chainconstant region in which the CH2 domain contains a D265A substitution(heavy chain amino acid numbering according to Kabat et al. (supra))results in a complete loss of interaction between the variant constantregion and IgG Fc receptors FcγRIIB, FcγRIII, FcγRI, and FcγRIV. Shieldset al. (2001) at page 6595, Table 1. See also Baudino et al. (2008) JImmunol 181:6664-6669 (supra).

Changes within the hinge region also affect effector functions. Forexample, deletion of the hinge region may reduce affinity for Fcreceptors and may reduce complement activation (Klein et al. (1981) ProcNatl Acad Sci USA 78: 524-528). The present disclosure therefore alsorelates to antibodies with alterations in the hinge region.

In some embodiments, an anti-C5 antibody may contain an altered constantregion exhibiting enhanced or reduced complement dependent cytotoxicity(CDC). Modulated CDC activity may be achieved by introducing one or moreamino acid substitutions, insertions, or deletions in an Fc region ofthe antibody. See, e.g., U.S. Pat. No. 6,194,551. Alternatively oradditionally, cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved or reducedinternalization capability and/or increased or decreasedcomplement-mediated cell killing. See, e.g., Caron et al. (1992) J ExpMed 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCTpublication nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988)Nature 322:738-40; and U.S. Pat. Nos. 5,648,260 and 5,624,821.

Another potential means of modulating effector function of antibodiesincludes changes in glycosylation, which is summarized in, e.g., Raju(2003) BioProcess International 1(4):44-53. According to Wright andMorrison, the microheterogeneity of human IgG oligosaccharides canaffect biological functions such as CDC and ADCC, binding to various Fcreceptors, and binding to C1q protein. (1997) TIBTECH 15:26-32.Glycosylation patterns of antibodies can differ depending on theproducing cell and the cell culture conditions (Raju, supra). Suchdifferences can lead to changes in both effector function andpharmacokinetics. See, e.g., Israel et al. (1996) Immunology89(4):573-578; and Newkirk et al. (1996) Clin Exp Immunol106(2):259-264. Differences in effector function may be related to theIgG's ability to bind to the Fcγ receptors (FcγRs) on the effectorcells. Shields et al. have shown that IgG, with alterations in aminoacid sequence that have improved binding to FcγR, can exhibit up to 100%enhanced ADCC using human effector cells. (2001) J Biol Chem276(9):6591-6604. While these alterations include changes in amino acidsnot found at the binding interface, both the nature of the sugarcomponent as well as its structural pattern may also contribute to thedifferences observed. In addition, the presence or absence of fucose inthe oligosaccharide component of an IgG can improve binding and ADCC.See, e.g., Shields et al. (2002) J Biol Chem 277(30):26733-26740. An IgGthat lacked a fucosylated carbohydrate linked to Asn²⁹⁷ exhibited normalreceptor binding to the FcγRI receptor. In contrast, binding to theFcγRIIIA receptor was improved 50-fold and accompanied by enhanced ADCC,especially at lower antibody concentrations.

Still other approaches exist for altering the effector function ofantibodies. For example, antibody-producing cells can be hypermutagenic,thereby generating antibodies with randomly altered polypeptide residuesthroughout an entire antibody molecule. See, e.g., PCT publication no.WO 05/011735. Hypermutagenic host cells include cells deficient in DNAmismatch repair. Antibodies produced in this manner may be lessantigenic and/or have beneficial pharmacokinetic properties.Additionally, such antibodies may be selected for properties such asenhanced or decreased effector function(s). Additional details ofmolecular biology techniques useful for preparing an antibody orantigen-binding fragment thereof described herein are set forth below.

Recombinant Antibody Expression and Purification

The antibodies or antigen-binding fragments thereof described herein canbe produced using a variety of techniques known in the art of molecularbiology and protein chemistry. For example, a nucleic acid encoding oneor both of the heavy and light chain polypeptides of an antibody can beinserted into an expression vector that contains transcriptional andtranslational regulatory sequences, which include, e.g., promotersequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, transcriptionterminator signals, polyadenylation signals, and enhancer or activatorsequences. The regulatory sequences include a promoter andtranscriptional start and stop sequences. In addition, the expressionvector can include more than one replication system such that it can bemaintained in two different organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification.

Various modifications, e.g., substitutions, can be introduced into theDNA sequences encoding the heavy and/or light chain polypeptidesdescribed herein using standard methods known to those of skill in theart. For example, introduction of a histidine substitution at one ormore CDR positions of an antibody can be carried out using standardmethods, such as PCR-mediated mutagenesis, in which the mutatednucleotides are incorporated into the PCR primers such that the PCRproduct contains the desired mutations or site-directed mutagenesis. Asubstitution may be introduced into one or more of the CDR regions toincrease or decrease the K_(D) of the antibody for antigen, e.g., at pH7.4 or pH 6.0. Techniques in site-directed mutagenesis are well-known inthe art. See, e.g., Sambrook et al., supra.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain polypeptides from nucleic acids inmammalian cells. One class of vectors relies upon the integration of thedesired gene sequences into the host cell genome. Cells which havestably integrated DNA can be selected by simultaneously introducing drugresistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc NatlAcad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol ApplGenet 1:327). The selectable marker gene can be either linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection (Wigler et al. (1979) Cell 16:77). A second class ofvectors utilizes DNA elements which confer autonomously replicatingcapabilities to an extrachromosomal plasmid. These vectors can bederived from animal viruses, such as bovine papillomavirus (Sarver etal. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitablefor subsequent expression of the nucleic acid. The method ofintroduction is largely dictated by the targeted cell type, discussedbelow. Exemplary methods include CaPO₄ precipitation, liposome fusion,cationic liposomes, electroporation, viral infection, dextran-mediatedtransfection, polybrene-mediated transfection, protoplast fusion, anddirect microinjection.

Appropriate host cells for the expression of antibodies orantigen-binding fragments thereof include yeast, bacteria, insect,plant, and mammalian cells. Of particular interest are bacteria such asE. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris,insect cells such as SF9, mammalian cell lines (e.g., human cell lines),as well as primary cell lines.

In some embodiments, an antibody or fragment thereof can be expressedin, and purified from, transgenic animals (e.g., transgenic mammals).For example, an antibody can be produced in transgenic non-human mammals(e.g., rodents) and isolated from milk as described in, e.g., Houdebine(2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al.(2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J ImmunolMethods 231(1-2):147-157.

The antibodies and fragments thereof can be produced from the cells byculturing a host cell transformed with the expression vector containingnucleic acid encoding the antibodies or fragments, under conditions, andfor an amount of time, sufficient to allow expression of the proteins.Such conditions for protein expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. For example,antibodies expressed in E. coli can be refolded from inclusion bodies(see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expressionsystems and methods for their use are well known in the art (see CurrentProtocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—ALaboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York(2001)). The choice of codons, suitable expression vectors and suitablehost cells will vary depending on a number of factors, and may be easilyoptimized as needed. An antibody (or fragment thereof) described hereincan be expressed in mammalian cells or in other expression systemsincluding but not limited to yeast, baculovirus, and in vitro expressionsystems (see, e.g., Kaszubska et al. (2000) Protein Expression andPurification 18:213-220).

Following expression, the antibodies and fragments thereof can beisolated. The term “purified” or “isolated” as applied to any of theproteins (antibodies or fragments) described herein refers to apolypeptide that has been separated or purified from components (e.g.,proteins or other naturally-occurring biological or organic molecules)which naturally accompany it, e.g., other proteins, lipids, and nucleicacid in a prokaryote expressing the proteins. Typically, a polypeptideis purified when it constitutes at least 60 (e.g., at least 65, 70, 75,80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in asample.

An antibody or fragment thereof can be isolated or purified in a varietyof ways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography. For example, an antibody can bepurified using a standard anti-antibody column (e.g., a protein-A orprotein-G column). Ultrafiltration and diafiltration techniques, inconjunction with protein concentration, are also useful. See, e.g.,Scopes (1994) “Protein Purification, 3^(rd) edition,” Springer-Verlag,New York City, N.Y. The degree of purification necessary will varydepending on the desired use. In some instances, no purification of theexpressed antibody or fragments thereof will be necessary.

Methods for determining the yield or purity of a purified antibody orfragment thereof are known in the art and include, e.g., Bradford assay,UV spectroscopy, Biuret protein assay, Lowry protein assay, amido blackprotein assay, high pressure liquid chromatography (HPLC), massspectrometry (MS), and gel electrophoretic methods (e.g., using aprotein stain such as Coomassie Blue or colloidal silver stain).

In some embodiments, endotoxin can be removed from the antibodies orfragments. Methods for removing endotoxin from a protein sample areknown in the art. For example, endotoxin can be removed from a proteinsample using a variety of commercially available reagents including,without limitation, the ProteoSpin™ Endotoxin Removal Kits (NorgenBiotek Corporation), Detoxi-Gel Endotoxin Removal Gel (ThermoScientific; Pierce Protein Research Products), MiraCLEAN® EndotoxinRemoval Kit (Mirus), or Acrodisc™—Mustang® E membrane (PallCorporation).

Methods for detecting and/or measuring the amount of endotoxin presentin a sample (both before and after purification) are known in the artand commercial kits are available. For example, the concentration ofendotoxin in a protein sample can be determined using the QCL-1000Chromogenic kit (BioWhittaker) or the limulus amebocyte lysate(LAL)-based kits such as the Pyrotell®, Pyrotell®-T, Pyrochrome®,Chromo-LAL, and CSE kits available from the Associates of Cape CodIncorporated.

Modification of the Antibodies or Antigen-Binding Fragments Thereof

The antibodies or antigen-binding fragments thereof can be modifiedfollowing their expression and purification. The modifications can becovalent or non-covalent modifications. Such modifications can beintroduced into the antibodies or fragments by, e.g., reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Suitable sites for modification can be chosen using any of avariety of criteria including, e.g., structural analysis or amino acidsequence analysis of the antibodies or fragments.

In some embodiments, the antibodies or antigen-binding fragments thereofcan be conjugated to a heterologous moiety. The heterologous moiety canbe, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxinor a drug), or a detectable label such as, but not limited to, aradioactive label, an enzymatic label, a fluorescent label, a heavymetal label, a luminescent label, or an affinity tag such as biotin orstreptavidin. Suitable heterologous polypeptides include, e.g., anantigenic tag (e.g., FLAG (DYKDDDDK (SEQ ID NO:20)), polyhistidine(6-His; HHHHHH (SEQ ID NO:21)), hemagglutinin (HA; YPYDVPDYA (SEQ IDNO:22)), glutathione-S-transferase (GST), or maltose-binding protein(MBP)) for use in purifying the antibodies or fragments. Heterologouspolypeptides also include polypeptides (e.g., enzymes) that are usefulas diagnostic or detectable markers, for example, luciferase, afluorescent protein (e.g., green fluorescent protein (GFP)), orchloramphenicol acetyl transferase (CAT). Suitable radioactive labelsinclude, e.g., ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Suitablefluorescent labels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488,phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700,Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any ofa variety of luminescent lanthanide (e.g., europium or terbium)chelates. For example, suitable europium chelates include the europiumchelate of diethylene triamine pentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labelsinclude, e.g., alkaline phosphatase, CAT, luciferase, and horseradishperoxidase.

Two proteins (e.g., an antibody and a heterologous moiety) can becross-linked using any of a number of known chemical cross linkers.Examples of such cross linkers are those which link two amino acidresidues via a linkage that includes a “hindered” disulfide bond. Inthese linkages, a disulfide bond within the cross-linking unit isprotected (by hindering groups on either side of the disulfide bond)from reduction by the action, for example, of reduced glutathione or theenzyme disulfide reductase. One suitable reagent,4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene (SMPT),forms such a linkage between two proteins utilizing a terminal lysine onone of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated tothe amino acid backbone of the antibody. Alternatively, the radioactivelabel can be included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds tofree amino groups to form meta-iodophenyl (mIP) derivatives of relevantproteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) orchelate (e.g., to DOTA or DTPA) which is in turn bound to the proteinbackbone. Methods of conjugating the radioactive labels or largermolecules/chelates containing them to the antibodies or antigen-bindingfragments described herein are known in the art. Such methods involveincubating the proteins with the radioactive label under conditions(e.g., pH, salt concentration, and/or temperature) that facilitatebinding of the radioactive label or chelate to the protein (see, e.g.,U.S. Pat. No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an antibody) are known in the art ofprotein chemistry. For example, fluorophores can be conjugated to freeamino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines)of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP)ester moieties attached to the fluorophores. In some embodiments, thefluorophores can be conjugated to a heterobifunctional cross-linkermoiety such as sulfo-SMCC. Suitable conjugation methods involveincubating an antibody protein, or fragment thereof, with thefluorophore under conditions that facilitate binding of the fluorophoreto the protein. See, e.g., Welch and Redvanly (2003) “Handbook ofRadiopharmaceuticals: Radiochemistry and Applications,” John Wiley andSons (ISBN 0471495603).

In some embodiments, the antibodies or fragments can be modified, e.g.,with a moiety that improves the stabilization and/or retention of theantibodies in circulation, e.g., in blood, serum, or other tissues. Forexample, the antibody or fragment can be PEGylated as described in,e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al.(2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al.(2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (FreseniusKabi, Germany; see, e.g., Pavisić et al. (2010) Int J Pharm387(1-2):110-119). The stabilization moiety can improve the stability,or retention of, the antibody (or fragment) by at least 1.5 (e.g., atleast 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the antibodies or antigen-binding fragments thereofdescribed herein can be glycosylated. In some embodiments, an antibodyor antigen-binding fragment thereof described herein can be subjected toenzymatic or chemical treatment, or produced from a cell, such that theantibody or fragment has reduced or absent glycosylation. Methods forproducing antibodies with reduced glycosylation are known in the art anddescribed in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

Pharmaceutical Compositions and Formulations

The compositions described herein can be formulated as a pharmaceuticalsolution, e.g., for administration to a subject for the treatment orprevention of a complement-associated disorder. The pharmaceuticalcompositions will generally include a pharmaceutically acceptablecarrier. As used herein, a “pharmaceutically acceptable carrier” refersto, and includes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66:1-19).

The compositions can be formulated according to standard methods.Pharmaceutical formulation is a well-established art, and is furtherdescribed in, e.g., Gennaro (2000) “Remington: The Science and Practiceof Pharmacy,” 20^(th) Edition, Lippincott, Williams & Wilkins (ISBN:0683306472); Ansel et al. (1999) “Pharmaceutical Dosage Forms and DrugDelivery Systems,” 7^(th) Edition, Lippincott Williams & WilkinsPublishers (ISBN: 0683305727); and Kibbe (2000) “Handbook ofPharmaceutical Excipients American Pharmaceutical Association,” 3^(rd)Edition (ISBN: 091733096X). In some embodiments, a composition can beformulated, for example, as a buffered solution at a suitableconcentration and suitable for storage at 2-8° C. (e.g., 4° C.). In someembodiments, a composition can be formulated for storage at atemperature below 0° C. (e.g., −20° C. or −80° C.). In some embodiments,the composition can be formulated for storage for up to 2 years (e.g.,one month, two months, three months, four months, five months, sixmonths, seven months, eight months, nine months, 10 months, 11 months, 1year, 1½ years, or 2 years) at 2-8° C. (e.g., 4° C.). Thus, in someembodiments, the compositions described herein are stable in storage forat least 1 year at 2-8° C. (e.g., 4° C.).

The pharmaceutical compositions can be in a variety of forms. Theseforms include, e.g., liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends, in part, on the intended mode ofadministration and therapeutic application. For example, compositionscontaining a composition intended for systemic or local delivery can bein the form of injectable or infusible solutions. Accordingly, thecompositions can be formulated for administration by a parenteral mode(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscularinjection). “Parenteral administration,” “administered parenterally,”and other grammatically equivalent phrases, as used herein, refer tomodes of administration other than enteral and topical administration,usually by injection, and include, without limitation, intravenous,intranasal, intraocular, pulmonary, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intrapulmonary, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural, intracerebral, intracranial, intracarotid and intrasternalinjection and infusion (see below).

The compositions can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating a composition described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filtersterilization. Generally, dispersions are prepared by incorporating acomposition described herein into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods for preparation include vacuumdrying and freeze-drying that yield a powder of a composition describedherein plus any additional desired ingredient (see below) from apreviously sterile-filtered solution thereof. The proper fluidity of asolution can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prolonged absorption ofinjectable compositions can be brought about by including in thecomposition a reagent that delays absorption, for example, monostearatesalts, and gelatin.

The compositions described herein can also be formulated inimmunoliposome compositions. Such formulations can be prepared bymethods known in the art such as, e.g., the methods described in Epsteinet al. (1985) Proc Natl Acad Sci USA 82:3688; Hwang et al. (1980) ProcNatl Acad Sci USA 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in, e.g., U.S.Pat. No. 5,013,556.

In certain embodiments, compositions can be formulated with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Many methods for thepreparation of such formulations are known in the art. See, e.g., J. R.Robinson (1978) “Sustained and Controlled Release Drug DeliverySystems,” Marcel Dekker, Inc., New York.

In some embodiments, compositions can be formulated in a compositionsuitable for intrapulmonary administration (e.g., for administration viaan inhaler or nebulizer) to a mammal such as a human. Methods forformulating such compositions are well known in the art and describedin, e.g., U.S. Patent Application Publication No. 20080202513; U.S. Pat.Nos. 7,112,341 and 6,019,968; and PCT Publication Nos. WO 00/061178 andWO 06/122257, the disclosures of each of which are incorporated hereinby reference in their entirety. Dry powder inhaler formulations andsuitable systems for administration of the formulations are describedin, e.g., U.S. Patent Application Publication No. 20070235029, PCTPublication No. WO 00/69887; and U.S. Pat. No. 5,997,848. Additionalformulations suitable for intrapulmonary administration (as well asmethods for formulating polypeptides) are set forth in, e.g., U.S.Patent Application Publication Nos. 20050271660 and 20090110679.

In some embodiments, compositions can be formulated for delivery to theeye. As used herein, the term “eye” refers to any and all anatomicaltissues and structures associated with an eye. The eye has a wallcomposed of three distinct layers: the outer sclera, the middle choroidlayer, and the inner retina. The chamber behind the lens is filled witha gelatinous fluid referred to as the vitreous humor. At the back of theeye is the retina, which detects light. The cornea is an opticallytransparent tissue, which conveys images to the back of the eye. Thecornea includes one pathway for the permeation of drugs into the eye.Other anatomical tissue structures associated with the eye include thelacrimal drainage system, which includes a secretory system, adistributive system and an excretory system. The secretory systemcomprises secretors that are stimulated by blinking and temperaturechange due to tear evaporation and reflex secretors that have anefferent parasympathetic nerve supply and secrete tears in response tophysical or emotional stimulation. The distributive system includes theeyelids and the tear meniscus around the lid edges of an open eye, whichspread tears over the ocular surface by blinking, thus reducing dryareas from developing.

In some embodiments, compositions can be administered locally, forexample, by way of topical application or intravitreal injection. Forexample, in some embodiments, the compositions can be formulated foradministration by way of an eye drop.

The therapeutic preparation for treating the eye can contain one or moreactive agents in a concentration from about 0.01 to about 1% by weight,preferably from about 0.05 to about 0.5% in a pharmaceuticallyacceptable solution, suspension or ointment. The preparation willpreferably be in the form of a sterile aqueous solution containing,e.g., additional ingredients such as, but not limited to, preservatives,buffers, tonicity agents, antioxidants and stabilizers, nonionic wettingor clarifying agents, and viscosity-increasing agents.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include, e.g., boric acid, sodium and potassiumbicarbonate, sodium and potassium borates, sodium and potassiumcarbonate, sodium acetate, and sodium biphosphate, in amounts sufficientto maintain the pH at between about pH 6 and pH 8, and preferably,between about pH 7 and pH 7.5. Suitable tonicity agents are dextran 40,dextran 70, dextrose, glycerin, potassium chloride, propylene glycol,and sodium chloride.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, and thiourea. Suitable wetting andclarifying agents include polysorbate 80, polysorbate 20, poloxamer 282and tyloxapol. Suitable viscosity-increasing agents include dextran 40,dextran 70, gelatin, glycerin, hydroxyethylcellulose,hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, andcarboxymethylcellulose. The preparation can be administered topically tothe eye of the subject in need of treatment (e.g., a subject afflictedwith AMD) by conventional methods, e.g., in the form of drops, or bybathing the eye in a therapeutic solution, containing one or morecompositions.

In addition, a variety of devices have been developed for introducingdrugs into the vitreal cavity of the eye. For example, U.S. patentapplication publication no. 20020026176 describes apharmaceutical-containing plug that can be inserted through the sclerasuch that it projects into the vitreous cavity to deliver thepharmaceutical agent into the vitreous cavity. In another example, U.S.Pat. No. 5,443,505 describes an implantable device for introduction intoa suprachoroidal space or an avascular region for sustained release ofdrug into the interior of the eye. U.S. Pat. Nos. 5,773,019 and6,001,386 each disclose an implantable drug delivery device attachableto the scleral surface of an eye. The device comprises an inner corecontaining an effective amount of a low solubility agent covered by anon-bioerodible polymer that is permeable to the low solubility agent.During operation, the low solubility agent permeates the bioerodiblepolymer cover for sustained release out of the device. Additionalmethods and devices (e.g., a transscleral patch and delivery via contactlenses) for delivery of a therapeutic agent to the eye are described in,e.g., Ambati and Adamis (2002) Prog Retin Eye Res 21(2):145-151; Rantaand Urtti (2006) Adv Drug Delivery Rev 58(11):1164-1181; Barocas andBalachandran (2008) Expert Opin Drug Delivery 5(1):1-10(10); Gulsen andChauhan (2004) Invest Opthalmol Vis Sci 45:2342-2347; Kim et al. (2007)Ophthalmic Res 39:244-254; and PCT publication no. WO 04/073551, thedisclosures of which are incorporated herein by reference in theirentirety.

As described above, relatively high concentration compositions can bemade. For example, the compositions can be formulated at a concentrationof between about 10 mg/mL to 100 mg/mL (e.g., between about 9 mg/mL and90 mg/mL; between about 9 mg/mL and 50 mg/mL; between about 10 mg/mL and50 mg/mL; between about 15 mg/mL and 50 mg/mL; between about 15 mg/mLand 110 mg/mL; between about 15 mg/mL and 100 mg/mL; between about 20mg/mL and 100 mg/mL; between about 20 mg/mL and 80 mg/mL; between about25 mg/mL and 100 mg/mL; between about 25 mg/mL and 85 mg/mL; betweenabout 20 mg/mL and 50 mg/mL; between about 25 mg/mL and 50 mg/mL;between about 30 mg/mL and 100 mg/mL; between about 30 mg/mL and 50mg/mL; between about 40 mg/mL and 100 mg/mL; or between about 50 mg/mLand 100 mg/mL). In some embodiments, compositions can be formulated at aconcentration of greater than 5 mg/mL and less than 50 mg/mL. Methodsfor formulating a protein in an aqueous solution are known in the artand are described in, e.g., U.S. Pat. No. 7,390,786; McNally and Hastedt(2007), “Protein Formulation and Delivery,” Second Edition, Drugs andthe Pharmaceutical Sciences, Volume 175, CRC Press; and Banga (2005),“Therapeutic peptides and proteins: formulation, processing, anddelivery systems, Second Edition” CRC Press. In some embodiments, theaqueous solution has a neutral pH, e.g., a pH between, e.g., 6.5 and 8(e.g., between and inclusive of 7 and 8). In some embodiments, theaqueous solution has a pH of about 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the aqueoussolution has a pH of greater than (or equal to) 6 (e.g., greater than orequal to 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, or 7.9), but less than pH 8.

Nucleic acids encoding a therapeutic polypeptide can be incorporatedinto a gene construct to be used as a part of a gene therapy protocol todeliver nucleic acids that can be used to express and produce agentswithin cells. Expression constructs of such components may beadministered in any therapeutically effective carrier, e.g. anyformulation or composition capable of effectively delivering thecomponent gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, lentivirus, and herpes simplexvirus-1 (HSV-1), or recombinant bacterial or eukaryotic plasmids. Viralvectors can transfect cells directly; plasmid DNA can be delivered withthe help of, for example, cationic liposomes (lipofectin) orderivatized, polylysine conjugates, gramicidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation (see, e.g.,WO04/060407) carried out in vivo. (See also, “Ex vivo Approaches,”below.) Examples of suitable retroviruses include pLJ, pZIP, pWE and pEMwhich are known to those skilled in the art (see, e.g., Eglitis et al.(1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc Natl AcadSci USA 85:6460-6464; Wilson et al. (1988) Proc Natl Acad Sci USA85:3014-3018; Armentano et al. (1990) Proc Natl Acad Sci USA87:6141-6145; Huber et al. (1991) Proc Natl Acad Sci USA 88:8039-8043;Ferry et al. (1991) Proc Natl Acad Sci USA 88:8377-8381; Chowdhury etal. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc NatlAcad Sci USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy3:641-647; Dai et al. (1992) Proc Natl Acad Sci USA 89:10892-10895; Hwuet al. (1993) J Immunol 150:4104-4115; U.S. Pat. Nos. 4,868,116 and4,980,286; and PCT Publication Nos. WO89/07136, WO89/02468, WO89/05345,and WO92/07573). Another viral gene delivery system utilizesadenovirus-derived vectors (see, e.g., Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in theart. Yet another viral vector system useful for delivery of the subjectgene is the adeno-associated virus (AAV). See, e.g., Flotte et al.(1992) Am J Respir Cell Mol Biol 7:349-356; Samulski et al. (1989) JVirol 63:3822-3828; and McLaughlin et al. (1989) J Virol 62:1963-1973.

In some embodiments, compositions can be formulated with one or moreadditional therapeutic agents, e.g., additional therapies for treatingor preventing a complement-associated disorder (e.g., an AP-associateddisorder or a CP-associated disorder) in a subject. Additional agentsfor treating a complement-associated disorder in a subject will varydepending on the particular disorder being treated, but can include,without limitation, an antihypertensive (e.g., an angiotensin-convertingenzyme inhibitor) [for use in treating, e.g., HELLP syndrome], ananticoagulant, a corticosteroid (e.g., prednisone), or animmunosuppressive agent (e.g., vincristine or cyclosporine A). Examplesof anticoagulants include, e.g., warfarin (Coumadin), aspirin, heparin,phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g.,argatroban, lepirudin, bivalirudin, or dabigatran). A compositiondescribed herein can also be formulated with a fibrinolytic agent (e.g.,ancrod, ε-aminocaproic acid, antiplasmin-a₁, prostacyclin, anddefibrotide) for the treatment of a complement-associated disorder. Insome embodiments, a composition can be formulated with a lipid-loweringagent such as an inhibitor of hydroxymethylglutaryl CoA reductase. Insome embodiments, a composition can be formulated with, or for use with,an anti-CD20 agent such as rituximab (Rituxan™; Biogen Idec, Cambridge,Mass.). In some embodiments, e.g., for the treatment of RA, thecomposition can be formulated with one or both of infliximab (Remicade®;Centocor, Inc.) and methotrexate (Rheumatrex®, Trexall®). In someembodiments, a composition described herein can be formulated with anon-steroidal anti-inflammatory drug (NSAID). Many different NSAIDS areavailable, some over the counter including ibuprofen (Advil®, Motrin®,Nuprin®) and naproxen (Alleve®) and many others are available byprescription including meloxicam (Mobic®), etodolac (Lodine®),nabumetone (Relafen®), sulindac (Clinoril®), tolementin (Tolectin®),choline magnesium salicylate (Trilasate®), diclofenac (Cataflam®,Voltaren®, Arthrotec®), Diflusinal (Dolobid®), indomethicin (Indocin®),Ketoprofen (Orudis®, Oruvail®), Oxaprozin (Daypro®), and piroxicam(Feldene®). In some embodiments a composition can be formulated for usewith an anti-hypertensive, an anti-seizure agent (e.g., magnesiumsulfate), or an anti-thrombotic agent. Anti-hypertensives include, e.g.,labetalol, hydralazine, nifedipine, calcium channel antagonists,nitroglycerin, or sodium nitroprussiate. (See, e.g., Mihu et al. (2007)J Gastrointestin Liver Dis 16(4):419-424.) Anti-thrombotic agentsinclude, e.g., heparin, antithrombin, prostacyclin, or low dose aspirin.

In some embodiments, compositions formulated for intrapulmonaryadministration can include at least one additional active agent fortreating a pulmonary disorder. The at least one active agent can be,e.g., an anti-IgE antibody (e.g., omalizumab), an anti-IL-4 antibody oran anti-IL-5 antibody, an anti-IgE inhibitor (e.g., montelukast sodium),a sympathomimetic (e.g., albuterol), an antibiotic (e.g., tobramycin), adeoxyribonuclease (e.g., Pulmozyme®), an anticholinergic drug (e.g.,ipratropium bromide), a corticosteroid (e.g., dexamethasone), aβ-adrenoreceptor agonist, a leukotriene inhibitor (e.g., zileuton), a5-lipoxygenase inhibitor, a PDE inhibitor, a CD23 antagonist, an IL-13antagonist, a cytokine release inhibitor, a histamine H1 receptorantagonist, an anti-histamine, an anti-inflammatory agent (e.g.,cromolyn sodium), or a histamine release inhibitor.

In some embodiments, compositions can be formulated for administrationwith one or more additional therapeutic agents for use in treating acomplement-associated disorder of the eye. Such additional therapeuticagents can be, e.g., bevacizumab or the Fab fragment of bevacizumab orranibizumab, both sold by Roche Pharmaceuticals, Inc., and pegaptanibsodium (Mucogen®; Pfizer, Inc.). Such a kit can also, optionally,include instructions for administering the composition to a subject.

In some embodiments, compositions can be formulated for administrationto a subject along with intravenous gamma globulin therapy (IVIG),plasmapheresis, plasma replacement, or plasma exchange. In someembodiments, compositions can be formulated for use before, during, orafter, a kidney transplant.

When compositions are to be used in combination with a second activeagent, the compositions can be coformulated with the second agent or thecompositions can be formulated separately from the second agentformulation. For example, the respective pharmaceutical compositions canbe mixed, e.g., just prior to administration, and administered togetheror can be administered separately, e.g., at the same or different times(see below).

Applications

The compositions described herein can be used in a number of diagnosticand therapeutic applications. For example, detectably-labeledantigen-binding molecules can be used in assays to detect the presenceor amount of the target antigens in a sample (e.g., a biologicalsample). The compositions can be used in in vitro assays for studyinginhibition of target antigen function. In some embodiments, e.g., inwhich the compositions bind to and inhibit a complement protein, thecompositions can be used as positive controls in assays designed toidentify additional novel compounds that inhibit complement activity orotherwise are useful for treating a complement-associated disorder. Forexample, a C5-inhibiting composition can be used as a positive controlin an assay to identify additional compounds (e.g., small molecules,aptamers, or antibodies) that reduce or abrogate C5 production orformation of MAC. The compositions can also be used in therapeuticmethods as elaborated on below.

Methods for Treatment

The compositions described herein can be administered to a subject,e.g., a human subject, using a variety of methods that depend, in part,on the route of administration. The route can be, e.g., intravenousinjection or infusion (IV), subcutaneous injection (SC), intraperitoneal(IP) injection, or intramuscular injection (IM).

Subcutaneous administration can be accomplished by means of a device.The device means may be a syringe, a prefilled syring, an auto-injectoreither disposable or reusable, a pen injector, a patch injector, awearable injector, an ambulatory syringe infusion pump with subcutaneousinfusion sets or other device for combining with the antibody drug forsubcutaneous injection.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures ofeach of which are incorporated herein by reference in their entirety. Acomposition described herein can be delivered to the subject by way ofan implantable device based on, e.g., diffusive, erodible, or convectivesystems, e.g., osmotic pumps, biodegradable implants, electrodiffusionsystems, electroosmosis systems, vapor pressure pumps, electrolyticpumps, effervescent pumps, piezoelectric pumps, erosion-based systems,or electromechanical systems.

In some embodiments, a composition described herein is therapeuticallydelivered to a subject by way of local administration. As used herein,“local administration” or “local delivery,” refers to delivery that doesnot rely upon transport of the composition or agent to its intendedtarget tissue or site via the vascular system. For example, thecomposition may be delivered by injection or implantation of thecomposition or agent or by injection or implantation of a devicecontaining the composition or agent. Following local administration inthe vicinity of a target tissue or site, the composition or agent, orone or more components thereof, may diffuse to the intended targettissue or site.

In some embodiments, a composition described herein can be locallyadministered to a joint (e.g., an articulated joint). For example, inembodiments where the disorder is arthritis, a therapeuticallyappropriate composition can be administered directly to a joint (e.g.,into a joint space) or in the vicinity of a joint. Examples ofintraarticular joints to which a composition described herein can belocally administered include, e.g., the hip, knee, elbow, wrist,sternoclavicular, temperomandibular, carpal, tarsal, ankle, and anyother joint subject to arthritic conditions. A composition describedherein can also be administered to bursa such as, e.g., acromial,bicipitoradial, cubitoradial, deltoid, infrapatellar, ischial, and anyother bursa known in the art of medicine.

In some embodiments, a composition described herein can be locallyadministered to the eye. As used herein, the term “eye” refers to anyand all anatomical tissues and structures associated with an eye. Theeye has a wall composed of three distinct layers: the outer sclera, themiddle choroid layer, and the inner retina. The chamber behind the lensis filled with a gelatinous fluid referred to as the vitreous humor. Atthe back of the eye is the retina, which detects light. The cornea is anoptically transparent tissue, which conveys images to the back of theeye. The cornea includes one pathway for the permeation of drugs intothe eye. Other anatomical tissue structures associated with the eyeinclude the lacrimal drainage system, which includes a secretory system,a distributive system and an excretory system. The secretory systemcomprises secretors that are stimulated by blinking and temperaturechange due to tear evaporation and reflex secretors that have anefferent parasympathetic nerve supply and secrete tears in response tophysical or emotional stimulation. The distributive system includes theeyelids and the tear meniscus around the lid edges of an open eye, whichspread tears over the ocular surface by blinking, thus reducing dryareas from developing.

In some embodiments, a composition described herein is administered tothe posterior chamber of the eye. In some embodiments, a compositiondescribed herein is administered intravitreally. In some embodiments, acomposition described herein is administered trans-sclerally.

In some embodiments, e.g., in embodiments for treatment or prevention ofa disorder such as COPD or asthma, a composition described herein can beadministered to a subject by way of the lung. Pulmonary drug deliverymay be achieved by inhalation, and administration by inhalation hereinmay be oral and/or nasal. Examples of pharmaceutical devices forpulmonary delivery include metered dose inhalers, dry powder inhalers(DPIs), and nebulizers. For example, a composition described herein canbe administered to the lungs of a subject by way of a dry powderinhaler. These inhalers are propellant-free devices that deliverdispersible and stable dry powder formulations to the lungs. Dry powderinhalers are well known in the art of medicine and include, withoutlimitation: the TurboHaler® (AstraZeneca; London, England) the AIR®inhaler (Alkermes®; Cambridge, Mass.); Rotahaler® (GlaxoSmithKline;London, England); and Eclipse™ (Sanofi-Aventis; Paris, France). Seealso, e.g., PCT Publication Nos. WO 04/026380, WO 04/024156, and WO01/78693. DPI devices have been used for pulmonary administration ofpolypeptides such as insulin and growth hormone. In some embodiments, acomposition described herein can be intrapulmonarily administered by wayof a metered dose inhaler. These inhalers rely on a propellant todeliver a discrete dose of a compound to the lungs. Examples ofcompounds administered by metered dose inhalers include, e.g., Astovent®(Boehringer-Ingelheim; Ridgefield, Conn.) and Flovent®(GlaxoSmithKline). See also, e.g., U.S. Pat. Nos. 6,170,717; 5,447,150;and 6,095,141.

In some embodiments, a composition described herein can be administeredto the lungs of a subject by way of a nebulizer. Nebulizers usecompressed air to deliver a compound as a liquefied aerosol or mist. Anebulizer can be, e.g., a jet nebulizer (e.g., air or liquid-jetnebulizers) or an ultrasonic nebulizer. Additional devices andintrapulmonary administration methods are set forth in, e.g., U.S.Patent Application Publication Nos. 20050271660 and 20090110679, thedisclosures of each of which are incorporated herein by reference intheir entirety.

In some embodiments, the compositions provided herein are present inunit dosage form, which can be particularly suitable forself-administration. A formulated product of the present disclosure canbe included within a container, typically, for example, a vial,cartridge, prefilled syringe or disposable pen. A doser such as thedoser device described in U.S. Pat. No. 6,302,855 may also be used, forexample, with an injection system of the present disclosure.

An injection system of the present disclosure may employ a delivery penas described in U.S. Pat. No. 5,308,341. Pen devices, most commonly usedfor self-delivery of insulin to patients with diabetes, are well knownin the art. Such devices can comprise at least one injection needle(e.g., a 31 gauge needle of about 5 to 8 mm in length), are typicallypre-filled with one or more therapeutic unit doses of a therapeuticsolution, and are useful for rapidly delivering the solution to asubject with as little pain as possible.

One medication delivery pen includes a vial holder into which a vial ofinsulin or other medication may be received. The vial holder is anelongate generally tubular structure with proximal and distal ends. Thedistal end of the vial holder includes mounting means for engaging adouble-ended needle cannula. The proximal end also includes mountingmeans for engaging a pen body which includes a driver and dose settingapparatus. A disposable medication (e.g., a high concentration solutionof a composition described herein) containing vial for use with theprior art vial holder includes a distal end having a pierceableelastomeric septum that can be pierced by one end of a double-endedneedle cannula. The proximal end of this vial includes a stopperslidably disposed in fluid tight engagement with the cylindrical wall ofthe vial. This medication delivery pen is used by inserting the vial ofmedication into the vial holder. A pen body then is connected to theproximal end of the vial holder. The pen body includes a dose settingapparatus for designating a dose of medication to be delivered by thepen and a driving apparatus for urging the stopper of the vial distallyfor a distance corresponding to the selected dose. The user of the penmounts a double-ended needle cannula to the distal end of the vialholder such that the proximal point of the needle cannula pierces theseptum on the vial. The patient then selects a dose and operates the pento urge the stopper distally to deliver the selected dose. The doseselecting apparatus returns to zero upon injection of the selected dose.The patient then removes and discards the needle cannula, and keeps themedication delivery pen in a convenient location for the next requiredmedication administration. The medication in the vial will becomeexhausted after several such administrations of medication. The patientthen separates the vial holder from the pen body. The empty vial maythen be removed and discarded. A new vial can be inserted into the vialholder, and the vial holder and pen body can be reassembled and used asexplained above. Accordingly, a medication delivery pen generally has adrive mechanism for accurate dosing and ease of use.

A dosage mechanism such as a rotatable knob allows the user toaccurately adjust the amount of medication that will be injected by thepen from a prepackaged vial of medication. To inject the dose ofmedication, the user inserts the needle under the skin and depresses theknob once as far as it will depress. The pen may be an entirelymechanical device or it may be combined with electronic circuitry toaccurately set and/or indicate the dosage of medication that is injectedinto the user. See, e.g., U.S. Pat. No. 6,192,891.

In some embodiments, the needle of the pen device is disposable and thekits include one or more disposable replacement needles. Pen devicessuitable for delivery of any one of the presently featured compositionsare also described in, e.g., U.S. Pat. Nos. 6,277,099; 6,200,296; and6,146,361, the disclosures of each of which are incorporated herein byreference in their entirety. A microneedle-based pen device is describedin, e.g., U.S. Pat. No. 7,556,615, the disclosure of which isincorporated herein by reference in its entirety. See also the PrecisionPen Injector (PPI) device, Molly™, manufactured by Scandinavian HealthLtd.

The present disclosure also presents controlled-release orextended-release formulations suitable for chronic and/orself-administration of a medication such as a composition describedherein. The various formulations can be administered to a patient inneed of treatment with the medication as a bolus or by continuousinfusion over a period of time.

In some embodiments, a high concentration composition described hereinis formulated for sustained-release, extended-release, timed-release,controlled-release, or continuous-release administration. In someembodiments, depot formulations are used to administer the compositionto the subject in need thereof. In this method, the composition isformulated with one or more carriers providing a gradual release ofactive agent over a period of a number of hours or days. Suchformulations are often based upon a degrading matrix which graduallydisperses in the body to release the active agent.

In some embodiments, a composition described herein is administered byway of intrapulmonary administration to a subject in need thereof. Forexample, a composition described herein can be delivered by way of anebulizer or an inhaler to a subject (e.g., a human) afflicted with adisorder such as asthma or COPD.

A suitable dose of a composition described herein, which dose is capableof treating or preventing a disorder in a subject, can depend on avariety of factors including, e.g., the age, sex, and weight of asubject to be treated and the particular inhibitor compound used. Forexample, a different dose of one composition (e.g., an anti-C5composition) may be required to treat a subject with RA as compared tothe dose of a different composition (e.g., an anti-TNFα composition)required to treat the same subject. Other factors affecting the doseadministered to the subject include, e.g., the type or severity of thedisorder. For example, a subject having RA may require administration ofa different dosage of an anti-C5 composition described herein than asubject with PNH. Other factors can include, e.g., other medicaldisorders concurrently or previously affecting the subject, the generalhealth of the subject, the genetic disposition of the subject, diet,time of administration, rate of excretion, drug combination, and anyother additional therapeutics that are administered to the subject. Itshould also be understood that a specific dosage and treatment regimenfor any particular subject will also depend upon the judgment of thetreating medical practitioner (e.g., doctor or nurse).

A composition described herein can be administered as a fixed dose, orin a milligram per kilogram (mg/kg) dose. In some embodiments, the dosecan also be chosen to reduce or avoid production of antibodies or otherhost immune responses against one or more of the antigen-bindingmolecules in the composition. While in no way intended to be limiting,exemplary dosages of an antibody, such as a composition described hereininclude, e.g., 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg,0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg. Exemplary dosages of acomposition described herein include, without limitation, 0.1 mg/kg, 0.5mg/kg, 1.0 mg/kg, 2.0 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg.

A pharmaceutical solution can include a therapeutically effective amountof a composition described herein. Such effective amounts can be readilydetermined by one of ordinary skill in the art based, in part, on theeffect of the administered composition, or the combinatorial effect ofthe composition and one or more additional active agents, if more thanone agent is used. A therapeutically effective amount of a compositiondescribed herein can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thecomposition (and one or more additional active agents) to elicit adesired response in the individual, e.g., amelioration of at least onecondition parameter, e.g., amelioration of at least one symptom of thecomplement-mediated disorder. For example, a therapeutically effectiveamount of a composition described herein can inhibit (lessen theseverity of or eliminate the occurrence of) and/or prevent a particulardisorder, and/or any one of the symptoms of the particular disorderknown in the art or described herein. A therapeutically effective amountis also one in which any toxic or detrimental effects of the compositionare outweighed by the therapeutically beneficial effects.

Suitable human doses of any of the compositions described herein canfurther be evaluated in, e.g., Phase I dose escalation studies. See,e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718;Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; andHetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10):3499-3500.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose,” or similar terms used herein are intended to mean anamount of an agent (e.g., a composition described herein) that willelicit the desired biological or medical response (e.g., an improvementin one or more symptoms of a complement-associated disorder). In someembodiments, a pharmaceutical solution described herein contains atherapeutically effective amount of at least one of said compositions.In some embodiments, the solutions contain one or more compositions andone or more (e.g., two, three, four, five, six, seven, eight, nine, 10,or 11 or more) additional therapeutic agents such that the compositionas a whole is therapeutically effective. For example, a solution cancontain an anti-C5 composition described herein and an immunosuppressiveagent, wherein the composition and agent are each at a concentrationthat when combined are therapeutically effective for treating orpreventing a complement-associated disorder (e.g., acomplement-associated inflammatory disorder such as COPD, asthma,sepsis, or RA) in a subject.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the complement-mediated disordersdescribed herein). These procedures can be used, e.g., for determiningthe LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. A composition described hereinthat exhibits a high therapeutic index is preferred. While compositionsthat exhibit toxic side effects may be used, care should be taken todesign a delivery system that targets such compounds to the site ofaffected tissue and to minimize potential damage to normal cells and,thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of thecomposition described herein lies generally within a range ofcirculating concentrations of the compositions that include the ED₅₀with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For a composition described herein, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe antibody which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. Insome embodiments, e.g., where local administration (e.g., to the eye ora joint) is desired, cell culture or animal modeling can be used todetermine a dose required to achieve a therapeutically effectiveconcentration within the local site.

In some embodiments, the methods can be performed in conjunction withother therapies for complement-associated disorders. For example, thecomposition can be administered to a subject at the same time, prior to,or after, plasmapheresis, IVIG therapy, or plasma exchange. See, e.g.,Appel et al. (2005) J Am Soc Nephrol 16:1392-1404. In some embodiments,the composition can be administered to a subject at the same time, priorto, or after, a kidney transplant.

A “subject,” as used herein, can be any mammal. For example, a subjectcan be a human, a non-human primate (e.g., orangutan, gorilla, macaque,baboon, or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, acat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, or a mouse. Insome embodiments, the subject is an infant (e.g., a human infant).

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment.

The term “preventing” is art-recognized, and when used in relation to acondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive a composition described herein. Thus, prevention of acomplement-associated disorder such as asthma includes, for example,reducing the extent or frequency of coughing, wheezing, or chest pain ina population of patients receiving a prophylactic treatment relative toan untreated control population, and/or delaying the occurrence ofcoughing or wheezing in a treated population versus an untreated controlpopulation, e.g., by a statistically and/or clinically significantamount.

As described above, the compositions described herein (e.g., anti-C5compositions) can be used to treat a variety of complement-associateddisorders such as, but not limited to: rheumatoid arthritis (RA); lupusnephritis; ischemia-reperfusion injury; atypical hemolytic uremicsyndrome (aHUS); typical or infectious hemolytic uremic syndrome (tHUS);dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH);multiple sclerosis (MS); macular degeneration (e.g., age-related maculardegeneration (AMD)); hemolysis, elevated liver enzymes, and lowplatelets (HELLP) syndrome; sepsis; dermatomyositis; diabeticretinopathy; thrombotic thrombocytopenic purpura (TTP); spontaneousfetal loss; Pauci-immune vasculitis; epidermolysis bullosa; recurrentfetal loss; multiple sclerosis (MS); and traumatic brain injury. See,e.g., Holers (2008) Immunological Reviews 223:300-316 and Holers andThurman (2004) Molecular Immunology 41:147-152. In some embodiments, thecomplement-mediated disorder is a complement-mediated vascular disordersuch as, but not limited to, a cardiovascular disorder, myocarditis, acerebrovascular disorder, a peripheral (e.g., musculoskeletal) vasculardisorder, a renovascular disorder, a mesenteric/enteric vasculardisorder, revascularization to transplants and/or replants, vasculitis,Henoch-Schönlein purpura nephritis, systemic lupuserythematosus-associated vasculitis, vasculitis associated withrheumatoid arthritis, immune complex vasculitis, organ or tissuetransplantation, Takayasu's disease, capillary leak syndrome, dilatedcardiomyopathy, diabetic angiopathy, thoracic-abdominal aortic aneurysm,Kawasaki's disease (arteritis), venous gas embolus (VGE), and restenosisfollowing stent placement, rotational atherectomy, and percutaneoustransluminal coronary angioplasty (PTCA). (See, e.g., U.S. patentapplication publication no. 20070172483.) In some embodiments, thecomplement-associated disorder is myasthenia gravis, cold-agglutinindisease (CAD), paroxysmal cold hemoglobinuria (PCH), dermatomyositis,scleroderma, warm autoimmune hemolytic anemia, Graves' disease,Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus,autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura(ITP), Goodpasture syndrome, antiphospholipid syndrome (APS), Degosdisease, and catastrophic APS (CAPS).

In some embodiments, a composition described herein, alone or incombination with a second anti-inflammatory agent, can be used to treatan inflammatory disorder such as, but not limited to, RA (above),inflammatory bowel disease, sepsis (above), septic shock, acute lunginjury, disseminated intravascular coagulation (DIC), or Crohn'sdisease. In some embodiments, the second anti-inflammatory agent can beone selected from the group consisting of NSAIDs, corticosteroids,methotrexate, hydroxychloroquine, anti-TNF agents such as etanercept andinfliximab, a B cell depleting agent such as rituximab, an interleukin-1antagonist, or a T cell costimulatory blocking agent such as abatacept.

In some embodiments, the complement-associated disorder is acomplement-associated neurological disorder such as, but not limited to,amyotrophic lateral sclerosis (ALS), brain injury, Alzheimer's disease,and chronic inflammatory demyelinating neuropathy.

Complement-associated disorders also include complement-associatedpulmonary disorders such as, but not limited to, asthma, bronchitis, achronic obstructive pulmonary disease (COPD), an interstitial lungdisease, α-1 anti-trypsin deficiency, emphysema, bronchiectasis,bronchiolitis obliterans, alveolitis, sarcoidosis, pulmonary fibrosis,and collagen vascular disorders.

In some embodiments, a composition described herein is administered to asubject to treat, prevent, or ameliorate at least one symptom of acomplement-associated inflammatory response (e.g., thecomplement-associated inflammatory response aspect of acomplement-associated disorder) in a subject. For example, a compositioncan be used to treat, prevent, and/or ameliorate one or more symptomsassociated with a complement-associated inflammatory response such asgraft rejection/graft-versus-host disease (GVHD), reperfusion injuries(e.g., following cardiopulmonary bypass or a tissue transplant), andtissue damage following other forms of traumatic injury such as a burn(e.g., a severe burn), blunt trauma, spinal injury, or frostbite. See,e.g., Park et al. (1999) Anesth Analg 99(1):42-48; Tofukuji et al.(1998) J Thorac Cardiovasc Surg 116(6):1060-1068; Schmid et al. (1997)Shock 8(2):119-124; and Bless et al. (1999) Am J Physiol 276(1):L57-L63.

In some embodiments, a composition described herein can be administeredto a subject as a monotherapy. Alternatively, as described above, thecomposition can be administered to a subject as a combination therapywith another treatment, e.g., another treatment for acomplement-associated disorder or a complement-associated inflammatoryresponse. For example, the combination therapy can include administeringto the subject (e.g., a human patient) one or more additional agents(e.g., anti-coagulants, anti-hypertensives, or anti-inflammatory drugs(e.g., steroids)) that provide a therapeutic benefit to a subject whohas, or is at risk of developing, sepsis. In another example, thecombination therapy can include administering to the subject one or moreadditional agents (e.g., an anti-IgE antibody, an anti-IL-4 antibody, ananti-IL-5 antibody, or an anti-histamine) that provide therapeuticbenefit to a subject who has, is at risk of developing, or is suspectedof having a complement-associated pulmonary disorder such as COPD orasthma. In some embodiments, a composition and the one or moreadditional active agents are administered at the same time. In otherembodiments, the composition is administered first in time and the oneor more additional active agents are administered second in time. Insome embodiments, the one or more additional active agents areadministered first in time and the composition is administered second intime.

A composition described herein can replace or augment a previously orcurrently administered therapy. For example, upon treating with acomposition described herein, administration of the one or moreadditional active agents can cease or diminish, e.g., be administered atlower levels, e.g., lower levels of eculizumab following administrationof an anti-C5 composition described herein. In some embodiments,administration of the previous therapy can be maintained. In someembodiments, a previous therapy will be maintained until the level ofthe composition reaches a level sufficient to provide a therapeuticeffect. The two therapies can be administered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in adisorder (e.g., sepsis, severe burn, RA, lupus nephritis, Goodpasturesyndrome, or asthma), as defined herein, means evaluating the subjectfor a change in a disease parameter, e.g., an improvement in one or moresymptoms of a given disorder. The symptoms of many of the abovedisorders (e.g., complement-associated disorders) are well known in theart of medicine. In some embodiments, the evaluation is performed atleast one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, orat least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or atleast 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more,after an administration of a composition described herein. The subjectcan be evaluated in one or more of the following periods: prior tobeginning of treatment; during the treatment; or after one or moreelements of the treatment have been administered. Evaluation can includeevaluating the need for further treatment, e.g., evaluating whether adosage, frequency of administration, or duration of treatment should bealtered. It can also include evaluating the need to add or drop aselected therapeutic modality, e.g., adding or dropping any of thetreatments for a complement-associated disorder described herein.

The following examples are merely illustrative and should not beconstrued as limiting the scope of this disclosure in any way as manyvariations and equivalents will become apparent to those skilled in theart upon reading the present disclosure. All patents, patentapplications and publications cited herein are incorporated herein byreference in their entireties.

EXAMPLES Example 1. Half-Life of Eculizumab is a Combination of SeveralClearance Rates

The average half-life of eculizumab in PNH and aHUS patients receivingthe prescribed dosing regimen is approximately 11-12 days, whereas theexpected half-life for a humanized monoclonal antibody having an IgG2/4Fc is predicted to be similar to that of an antibody containing an IgG2or IgG4 Fc, approximately 21-28 days. Morell et al. (1970) J Clin Invest49(4):673-680. To understand the potential impact of antigen-mediatedclearance on the overall clearance rate of eculizumab, the followingexperiments were performed using the human neonatal Fc receptor (hFcRn)mouse model (the mice lack endogenous FcRn but are transgenic for hFcRn(B6.Cg-Fcgrt^(tm1Dcr) Tg(FCGRT)32Dcr/DcrJ; Stock Number 014565, JacksonLaboratories, Bar Harbor, Me.)). The transgenic FcRn model has beendescribed in, e.g., Petkova et al. (2006) Int Immunology18(12):1759-1769; Oiao et al. (2008) Proc Natl Acad Sci USA105(27):9337-9342; and Roopenian et al. (2010) Methods Mol Biol602:93-104.

A single dose of 100 μg of eculizumab in 200 μL of phosphate bufferedsaline (PBS) was administered by intravenous (i.v.) injection to each offive hFcRn transgenic mice. Blood samples of approximately 100 μL werecollected from each of the mice at days one, three, seven, 14, 21, 28,and 35 following the administration. The concentration of eculizumab inserum was measured by ELISA. Briefly, assay plates were coated with asheep anti-human Igκ light chain capture antibody and blocked. The wellsof the plate were then contacted with the serum samples under conditionsthat allow eculizumab, if present in the serum, to bind to the captureantibody. The relative amount of eculizumab bound to each well wasdetected using a detectably-labeled anti-human IgG4 antibody andquantified relative to a standard curve generated from naïve mouse serumcontaining known quantities of eculizumab.

Antibody serum half-life was calculated using the following formula:

${Halflife} = {T \times \frac{\ln \mspace{14mu} 2}{\ln \frac{A_{0}}{A_{t}}}}$

Where: T=Time elapsed, A_(o)=Original serum concentration of theantibody (concentration at day 1 in the present study) and A_(t)=Amountof the antibody remaining after elapsed time T (minimal detectableconcentration or the last bleeding time point (day 35) in the presentstudy).

The results of the experiment are depicted in FIG. 1. The half-life ofeculizumab in the hFcRn mouse model was 13.49±0.93 days.

To determine the effect of human C5 on the half-life of eculizumab usingthe hFcRn model, antibody was premixed with a 4:1 molar ratio of humanC5 (Complement Technology Inc., Catalog Number: A120) prior to dosing, Adose of 100 μg of eculizumab was intravenously (i.v.) administered onday 0. Approximately 100 μL blood was collected into 1.5 mL Eppendorftubes for serum via retro-orbital bleeding at 1, 3, 7, 14, 21, 28 and 35days.

As shown in FIG. 1, the half-life of eculizumab in the hFcRn mouse modelin the presence of C5 was 4.55±1.02 days. These results indicate that,in addition to endocytosis-mediated antibody clearance mechanisms inwhich a long half-life is governed largely by FcRn-mediated recycling,the half-life of eculizumab may be significantly impacted byantigen-mediated clearance through human C5.

Example 2. Amino Acid Substitutions in the Fc Domain of EculizumabIncrease the Half-Life of Eculizumab but are not Sufficient to Overcomethe Effect of C5 on Eculizumab Clearance

Certain amino acid substitutions in the Fc region of an IgG antibodyhave been shown to lessen the rate of elimination of the antibody fromcirculation. Substitutions that increase the binding affinity of an IgGantibody for FcRn at pH 6.0 are examples of such a biological effect.See, e.g., Dall'Acqua et al. (2006) J Immunol 117:1129-1138 and Ghetieet al. (1997) Nat Biotech 15: 637-640. Zalevsky et al. [(2010) NatBiotech 28:157-159] describe a number of amino acid substitutions, e.g.,M428L/N434S, capable of increasing the half-life of an IgG antibody inserum. Other half-life extending amino acid substitutions include, e.g.,T250Q/M428L and M252Y/S254T/T256E. See, e.g., International patentapplication publication no. WO 2008/048545 and Dall'Acqua et al. (2006)J Biol Chem 281:23514-23524. To determine whether one or more amino acidsubstitutions in the Fc constant region of eculizumab are capable ofextending the half-life of eculizumab in serum, the followingsubstitutions were introduced into eculizumab: M252Y/S254T/T256E, basedon the EU numbering index (herein after this variant of eculizumab isreferred to as the YTE variant). The heavy chain constant regionconsisted of the following amino acid sequence:

(SEQ ID NO: 15) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.The amino acid sequence for the full-length heavy chain polypeptide ofthe YTE variant of eculizumab is depicted in SEQ ID NO:16.

The YTE variant was evaluated alongside eculizumab in the hFcRn mousemodel described in Example 1. That is, 100 μg of eculizumab (IgG2/4 Fcregion), a variant of eculizumab containing an Fc or the YTE variant ofeculizumab in 200 μL of phosphate buffered saline (PBS) was administeredby intravenous (i.v.) injection to each of eight hFcRn transgenic mice.Serum was collected from each of the mice at days one, three, seven, 14,21, 28, and 35 following the administration. The concentration of eachantibody in the serum was measured by ELISA and the half-life calculatedas described in Example 1. The results are depicted in FIG. 2 and Table2.

TABLE 2 Standard Error Antibody Tested Half-Life (SE) Eculizumab 13.490.93 Eculizumab-IgG2 14.28 1 Eculizumab-IgG2- 29.07 4.7 YTE

As shown in FIG. 2 and Table 2, the YTE substitution increased the meanhalf-life of eculizumab more than 2-fold from 14.28±1 days to 29.07±4.7days.

To determine the effect of human C5 on the half-life of the YTE variantof eculizumab, mice were administered human C5 as described above inExample 1. A dose of 100 μg of eculizumab, the eculizumab-IgG2 variant,or the eculizumab-IgG2 YTE variant was intravenously administered on day0. As shown in FIG. 3 and Table 3, the half-life of eculizumab, theeculizumab-IgG2 variant, and the eculizumab-IgG2 YTE variant decreasedsignificantly in the presence of a molar excess of human C5. Thus, aminoacid substitutions in FcRn-binding domain of eculizumab wereinsufficient to overcome the contribution of C5-mediated clearance onthe half-life of eculizumab.

TABLE 3 Standard Antibody Tested T½ Error (SE) Eculizumab 13.49 0.93Eculizumab-IgG2 14.28 1 Eculizumab-IgG2(YTE) 29.07 4.7 Eculizumab + hC54.55 1.02 Eculizumab-IgG2 + C5 2.11 0.31 Eculizumab-IgG2(YTE) + hC5 4.281.09

Example 3. The Effect of Amino Acid Substitutions in the CDRs ofEculizumab on Half-Life

As described above, the half-life of eculizumab in mice is significantlyshorter in the presence of its antigen, human C5 (hC5). While not beingbound by any particular theory or mechanism of action, it ishypothesized that the accelerated clearance in the presence of antigenis, in part, the result of the very high affinity of eculizumab for C5(K_(D)˜30 pM at pH 7.4 and ˜600 pM at pH 6.0) which does not allowefficient dissociation of the antibody:C5 complex in the early endosomalcompartments after pinocytosis. Without dissociation, theantibody:antigen complex is either recycled to the extracellularcompartment via the neonatal Fc receptor (FcRn) or targeted forlysosomal degradation. In either case the antibody is incapable ofbinding more than two C5 molecules in its lifetime.

The strong affinity of eculizumab for C5 (K_(D)˜30 pM) allows for nearcomplete binding of all C5 in blood, ensuring that very little C5 isactivated to form C5a and TCC. The affinity of eculizumab for C5 istherefore directly connected to the in vivo efficacy of the antibody inpatients treated with the antibody. The inventors set out to weaken theaffinity of eculizumab for C5, without compromising the efficacy ofeculizumab in vivo. While the disclosure is not limited to such anapproach, this was achieved by introducing histidine into one or morepositions in the CDRs of eculizumab. Histidine has a pKa of 6.04. Thismeans that as pH values drop from 7.4 (blood) to less than 6.0 (earlyendosomes), histidines gain a proton. Thus, in the endosome, histidinesbecome more positively-charged. The inventors hypothesized thatintroducing histidines at or near the binding site for C5 in eculizumab,the charge shift in the endosome may disrupt binding in the endosome,whilst preserving the high affinity for C5 at neutral pH in the blood.Such substitutions are hypothesized to increase the half-life byfacilitating the dissociation of antibody from the antibody:C5 complexin the acidic environment of the endosome, allowing free antibody to berecycled while the C5 is degraded in the lysosome.

Using eculizumab as the parent antibody, a series of variant antibodieswas generated in which every CDR position was substituted with ahistidine. The heavy chain variable region of eculizumab has thefollowing amino acid sequence:

(SEQ ID NO: 7) QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.(The CDR regions of the heavy chain variable region are underlined.) Thelight chain variable region of eculizumab has the following amino acidsequence:

(SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

The result of this histidine-scanning effort was 66 single histidinesubstitution variants of eculizumab. The light chain and heavy chaincoding sequences for these antibody variants were cloned into separate“single gene construct” plasmids suitable for expression in mammaliancells and sequence confirmed. Antibodies containing a single amino acidsubstitution were expressed transiently in HEK293F cells byco-transfection of single gene constructs encoding a single light chainor heavy chain. A co-transfection of “wildtype” heavy and light chainsrepresenting unmodified eculizumab CDR sequences was also performed(EHL000). Tissue culture supernatants were normalized for antibodyexpression level and use to evaluate antibody binding to human C5,relative to EHL000, using biolayer interferometry on an Octet Redinstrument (ForteBio Inc.). Briefly, antibodies were captured on ananti-human IgG Fc biosensor (ForteBio, cat #18-5001). Loaded tips werethen exposed to a pH 7.4 buffered solution containing 12.5 nM of nativepurified human C5 for 800 seconds to assess the kinetics of associationrelative to the parental antibody. Dissociation kinetics were assessedby transferring the tip to a pH 7.4 buffered solution or pH 6.0 bufferedsolution for 800 seconds. All measurements were repeated to ensureconsistency of readings.

Single histidine substitution variants of eculizumab were selected basedon a series of three properties relative to eculizumab. Preferredhistidine variants only deviated from the k_(a) and k_(d) of eculizumabat pH 7.4 to a minor degree, but deviated from the k_(d) of eculizumabat pH 6.0 more significantly. The relative threshold selection criteriawere as follows:

-   -   (1) a maximum variation for association kinetics at pH 7.4 of a        33% smaller peak phase shift at 800 seconds as compared to the        averaged peak phase shift at 800 seconds observed for        eculizumab;    -   (2) a maximum variation for dissociation kinetics at pH 7.4 of        no more than 3-fold reduction in peak phase shift over 800        seconds as compared to the averaged peak phase shift at 800        seconds observed for eculizumab; and    -   (3) a minimum variation for dissociation kinetics at pH 6.0 of        at least a 3-fold reduction in the peak phase shift over 800        seconds as compared to the averaged peak phase shift at 800        seconds observed for eculizumab.        For example, with respect to prong (1) above, if the average        peak phase shift after 800 seconds of association with        eculizumab is approximately 0.75 nm, a test antibody that has a        phase shift of less than 0.5 nm (e.g., reproduced two or more        times) would not meet the above criteria. By contrast, a test        antibody with greater than a 0.5 nm peak phase shift at 800        seconds meets the first criterion.

Single substitutions in the light chain variable region that met thesethresholds were the following: G31H, L33H, V91H, and T94H, all relativeto SEQ ID NO:8. Single substitutions in the heavy chain variable regionthat met these thresholds were the following: Y27H, I34H, L52H, andS57H, all relative to SEQ ID NO:7. See FIGS. 5A, 5B, 5C, and 5D.

A second series of antibodies was generated containing all possiblecombinations of two histidine substitutions at positions where singlesubstitutions met threshold criteria. See Table 1. These association anddissociation kinetics were analyzed via the same methods and compared toboth the original parental antibody and the single histidinesubstitutions. Likewise, a third and fourth series of antibodiescontaining three or four histidine substitutions, respectively, weregenerated and association and dissociation kinetics were analyzedcompared to the relevant two or three histidine substitutionpredecessors. See Table 1. At each stage the same criteria were used forminimum thresholds for association kinetics at pH 7.4, maximumthresholds for dissociation kinetics at pH 7.4 and minimum thresholdsfor dissociation kinetics at pH 6. Eight substitution combinations metthe above criteria and selected for affinity determination at pH 7.4 andpH 6.0 via SPR. The affinities are set forth in Table 4.

TABLE 4 ratio of KD pH KD pH KD at Clone VL VH 7.4 6.0 pH 6.0/pHDesignation Sequence Sequence (nM) (nM) 7.4 eculizumab SIN: 8 SIN: 70.033 0.685 21 EHL000 SIN: 8 SIN: 7 0.018 0.419 24 EHL001 G31H, SIN: 70.330 1900 5758 relative to SIN: 8. EHL004 G31H, S57H 0.135 374 2770relative to SIN: 8. EHL046 G31H, SIN: 7, 1.150 ND NA relative with: toSIN: 8. Y27H, L52H EHL049 G31H, SIN: 7, 0.573 ND NA relative with: toSIN: 8. Y27H, S57H EHL055 G31H, SIN: 7, 0.623 2550 4093 relative with:to SIN: 8. I34H, S57H EHG302 SIN: 8 SIN: 7, 0.289 10.0 35 with: Y27H,L52H EHG303 SIN: 8 SIN: 7, 0.146 1190 8151 with: Y27H, S57H EHG305 SIN:8 SIN: 7, 0.160 10.8 68 with: I34H, S57H *SIN refers to SEQ ID NO.

For these combinations of substitutions, the affinity of eculizumab forC5 was reduced by greater than 1000 fold at pH 6.0, while the affinitysuffered no greater than a 20-fold reduction in affinity at pH 7.0. Fromthese, EHG303 (Table 4) was selected for further analysis due to itshigh affinity at pH 7.4 (0.146 nM) and the ratio of (K_(D) at pH6.0)/(K_(D) at pH 7.4) of over 8,000. The heavy chain polypeptide of theEHG303 antibody comprises the following amino acid sequence:

(SEQ ID NO: 24) MGWSCIILFLVATATGVHS LEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK.The light chain polypeptide of the EHG303 antibody comprises thefollowing amino acid sequence:

(SEQ ID NO: 25) MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In the above sequences, the underlined portions correspond to the leadersequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

Also selected was the EHL049 antibody. Its heavy chain polypeptidecomprises the following amino acid sequence:

(SEQ ID NO: 26) MGWSCIILFLVATATGVHS LEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK.The light chain polypeptide of the EHL049 antibody comprises thefollowing amino acid sequence:

(SEQ ID NO: 27) MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In the above sequences, the underlined portions correspond to the leadersequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

Finally, the EHL000 heavy chain polypeptide comprises the followingamino acid sequence:

(SEQ ID NO: 28) MGWSCIILFLVATATGVHS LEQVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK.The light chain polypeptide of the EHL000 antibody comprises thefollowing amino acid sequence:

MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In the above sequences, the underlined portions correspond to the leadersequence of each polypeptide and the italicized portions areheterologous amino acids introduced by virtue of cloning.

Example 4. Histidine Substitutions Prolong the Half-Life of Eculizumabin Serum

The light chain polypeptide and heavy chain polypeptide of each of theEHL and EHG antibodies above, were expressed from single geneconstructs. Heavy and light chain coding sequences from EHG303 werecombined into a double gene expression vector, as were the light andheavy chain sequences for the EHL049 antibody. The resulting EHG303clone was designated as BNJ421 and the resulting EHL049 clone wasdesignated as BNJ423. The amino acid sequence of the heavy chainvariable region of BNJ421 is as follows:

(SEQ ID NO: 12) QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWMGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.The light chain variable region amino acid sequence for BNJ421 is asfollows:

(SEQ ID NO: 8) DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.The heavy chain variable region of the BNJ423 antibody comprises thefollowing amino acid sequence:

(SEQ ID NO: 12) QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWMGEILPGSG H TEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS.The light chain amino acid sequence for BNJ423 is as follows:

(SEQ ID NO: 18) DIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQ GTKVEIK.

These two molecules were evaluated alongside EHL000 in mice that wereimmunodeficient (NOD/scid) and C5 deficient. A single dose of 100 μg ofEHL000, BNJ421, or BNJ423 in 200 μL of phosphate buffered saline (PBS)was administered by intravenous (i.v.) injection to each of eight mice.Serum was collected from each of the mice at days one, three, seven, 14,21, 28, and 35 following the administration. The concentration of eachantibody in the serum was measured by ELISA. Antibody serum half-lifewas calculated using Pharsight Phoenix® WinNonlin® version 6.3 softwareby using the non-compartmental analysis (NCA) and direct response Emax.The percentage of the antibody remaining in the serum was calculated asfollows:

% of antibody remaining=C _(t) /C ₁×100

wherein, C_(t)=Antibody concentration on a given day; and C₁=Antibodyconcentration on day 1.The results are depicted in FIG. 6 and Table 5.

TABLE 5 Serum T ½ Standard Ab Tested (days) Error (SE) EHL000 22.18 1.01BNJ421 25.29 0.81 BNJ423 24.69 2.16

To determine the effect of human C5 on the half-life of these antibodiesusing the same mouse model, mice were administered human C5subcutaneously at a loading dose of 250 μg at day −1 (the day before theantibodies were administered to the mice), followed by twice daily dosesof 50 μg of C5 to maintain the serum C5 concentration at approximately20 μg/mL (as described in Example 1).

As shown in FIG. 7 (and Table 6, below), the half-life of EHL000(eculizumab-IgG1) in the mouse model in the presence of human (hC5) (ata concentration that was greater than a 1:1 molar ratio of C5 toeculizumab) was 2.49±0.34 days, whereas the half-life of the BNJ421 andBNJ423 antibodies (containing the histidine substitutions) wassubstantially greater at 15.25±0.90 days and 22.71±0.71 days,respectively. These results indicate that histidine substitutions in theCDRs of eculizumab, and the resultant pH-dependent affinity for C5,significantly decrease the rate of clearance of the eculizumab variantsfrom serum relative to eculizumab.

TABLE 6 Serum T ½ Ab Tested (days) SE EHL000 22.18 1.01 BNJ421 25.290.81 BNJ423 24.69 2.16 EHL000 + hC5 2.49 0.34 BNJ421 + hC5 15.25* 0.90BNJ423 + hC5 22.71 1.19 *Significant relative to EHL000 + hC5.

Example 5. Histidine Substituted-Eculizumab Variants do not LoseComplement-Inhibitory Activity

In addition, the serum hemolytic activity in each of the samplescontaining human C5 from the experiments described in Example 4 werealso evaluated. Terminal complement activity in mouse sera wasdetermined by assessing its ability to lyse chicken erythrocytes. Sincethe mice used were C5 deficient, the hemolytic activity directlyreflects the activity of human C5 in the sample. Briefly, antibodies at50, 3, and 0 μg/mL in Gelatin Veronal-Buffered Saline (GVBS) (ComptechCatalog # B100) containing 0.1% gelatin, 141 mM NaCl, 0.5 mM MgCl₂, 0.15mM CaCl₂, and 1.8 mM sodium barbital were used as low, medium and 100%lysis control, respectively. Experimental samples were prepared bydiluting the murine test serum 1:10 in GVBS. Sample aliquots (50 μL)were dispensed to corresponding triplicate wells of a 96-well plate(Corning; Tewksbury, Mass. Catalog #3799) containing an equal volume of20% mouse C5-deficient serum and 20% human serum (Bioreclamation,Catalog# HMSRM-COMP+) in GVBS in control wells and an equal volume of20% mouse C5-deficient serum and 20% human C5-depleted serum (ComplementTechnologies, Catalog number A320) in GVBS in test sample wells. EDTA (2μL at 500 mM, Sigma, catalog number E-9884) was added into the thirdwell of both control and sample triplicates to generate “no hemolysis”serum color control. Chicken erythrocytes were washed in GVBS,sensitized to activate the complement classical pathway by incubationwith an anti-chicken RBC polyclonal antibody (Intercell Technologies;0.1% v/v) at 4° C. for 15 minutes, washed again, and re-suspended inGVBS at a final concentration of ˜7.5×10⁷ cells/mL. The sensitizedchicken erythrocytes (˜2.5×10⁶ cells) were added to the plate containingthe controls and samples, mixed briefly on a plate shaker, and incubatedat 37° C. for 30 min. The reagents were mixed again, centrifuged at845×g for 3 min, and 85 μL of the supernatant was transferred to wellsof a 96-well flat-bottom microtiter plate (Nunc, Penfield, N.Y.,Catalog#439454). Absorbance was measured at 415 nm using a microplatereader and the percentage of hemolysis was determined using thefollowing formula:

${\% \mspace{14mu} {of}\mspace{14mu} {hemolysis}} = {\frac{\begin{matrix}{{{Sample}\mspace{14mu} {OD}} -} \\{{Sample}\mspace{14mu} {color}\mspace{14mu} {control}\mspace{20mu} {OD}}\end{matrix}}{\begin{matrix}{{100\% \mspace{14mu} {lysis}\mspace{14mu} {control}\mspace{14mu} {OD}} -} \\{100\% \mspace{14mu} {lysis}\mspace{14mu} {color}\mspace{14mu} {control}\mspace{14mu} {OD}}\end{matrix}} \times 100}$

-   -   where, OD=optical density.

As shown in FIG. 8, despite the slight reduction in affinity at pH 7.4relative to eculizumab, both BNJ421 and BNJ423 were still capable ofbinding nearly all of the human C5 present in circulation and inhibitinghemolysis. These results indicate the affinity of eculizumab for C5 canbe weakened without compromising the efficacy of the antibody in vivo,and conferring upon the antibody an increase serum half-life.

Example 6. pH-Dependent Binding to C5 and Enhanced FcRn-MediatedRecycling are Additive for Serum Half-Life of Eculizumab Variants

As shown above, in the presence of human C5, the half-life of ahistidine-substituted eculizumab variant was significantly extended intransgenic mice. To assess the potential additive effects ofpH-dependent binding to C5 and to FcRn on the pharmacokinetics (PK) andpharmacodynamics (PD) of anti-C5 antibodies in the presence ofconstitutive C5 synthesis and human FcRn, a series of PK/PD experimentswere performed using anti-mouse C5 antibodies with human constantregions in transgenic mice expressing human FcRn. These murine anti-C5antibodies were engineered from the variable region of BB5.1, a murineantibody that serves as a pharmacologic surrogate for eculizumab as itbinds mouse C5 and prevents its cleavage into the active metabolicfragments C5a and C5b [De Vries et al. (2003) Transplantation3:375-382]. A high affinity anti-mouse C5 antibody (designated as:BHL011) was engineered with an affinity-optimized variant of the BB5.1murine variable regions and human Igκ and human IgG2/G4 constantregions. A pH-dependent variant of BHL011 was engineered byincorporating three histidine substitutions into the murine variableregions (this variant was designated as: BHL006). A third antibody wasengineered by incorporating two amino acid substitutions into the humanconstant region heavy chain (M428L, N434S) to increase the affinity forhFcRn (this variant was designated as: BHL009).

The amino acid sequence of the light chain polypeptide of BHL006 is asfollows:

(SEQ ID NO: 30) NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.The amino acid sequence of the heavy chain polypeptide of the BHL006antibody is as follows:

(SEQ ID NO: 31) QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.The amino acid sequence of the light chain polypeptide of BHL009 is asfollows:

(SEQ ID NO: 32) NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.The amino acid sequence of the heavy chain polypeptide of BHL009 is asfollows:

(SEQ ID NO: 33) QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK.

The amino acid sequence of the light chain polypeptide of BHL011 is asfollows:

(SEQ ID NO: 34) NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQYLSSRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.The amino acid sequence of the heavy chain polypeptide of BHL011 is asfollows:

(SEQ ID NO: 35) QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The kinetics of BHL011, BHL006 and BHL009 binding to purified mouse weredetermined via SPR on a BIACore 3000 instrument using an anti-Fc humancapture method. Briefly, anti-human Fc (KPL, catalogue number: 01-10-20)diluted to 0.1 mg/mL in 10 mM sodium acetate pH 5.0, was immobilized ontwo flow cells of a CM5 chip for 8 minutes by amine coupling. Theantibodies were diluted to 0.25 μg/mL in running buffer (HBS-EP; 0.01 MHEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE LifeSciences, catalogue number: BR1001-88). The diluted antibody was theninjected on one flow cell followed by an injection of 6 nM mouse C5 onboth cells. The second flow cell was used as a reference surface. Thebinding was evaluated at pH 7.4 and pH 6.0. The surface was regeneratedeach time with 20 mM HCl, 0.01% P20. The data was processed with a 1:1Langmuir model using BIAevaluation 4.1 software with ‘doublereferencing’. The dissociation of BHL011, BHL006 and BHL009 complexed tomouse C5 at pH 6.0 were evaluated similarly, with an injection of 6 nMmouse C5 (pH 7.4) followed by an injection of HBS-EP buffer (pH 6.0).The results of these experiments are shown in Table 7.

TABLE 7 Dissociation Association Rate: Dissociation Rate: Constant: %Diss'n K_(a) (1/M*s) K_(d) (1/s) K_(D)(nM) Chi² in 300 sec Ab pH 7.4 pH6.0 pH 7.4 pH 6.0 pH 7.4 pH 6.0 pH 7.4 pH 6.0 pH 7.4 pH 6.0 BHL011 6.44× 10⁵ 2.39 × 10³ 6.13 × 10⁻⁵ 1.28 × 10⁻⁴ 0.0952 53.6 0.0194 0.048 1 7BHL006 2.93 × 10⁵ NB 1.02 × 10⁻³ NB 3.49 NB 0.021 NB 28 100 BHL009 2.61× 10⁵ NB 1.09 × 10⁻³ NB 4.19 NB 0.0234 NB 28 100 *“NB” no specificbinding was observed; “Ab” refers to antibody designation.

In order to determine the effects of pH-dependent binding to C5 on thepharmacokinetics (PK) of an anti-C5 antibody in the presence ofconstitutive C5 synthesis and the potential for enhanced FcRn recyclingto confer additive increases in half-life, the total serum concentrationof BHL011, BHL006 and BHL009 were analyzed using the transgenic FcRnmouse model described in Example 1. Total antibody serum concentrationand serum concentration as a percentage of the day 1 concentration areshown in FIGS. 9-11. Male mice are represented as solid lines andfemales as dashed lines. Total antibody serum concentrations at day 1were higher for females than for males, proportional to the differencesin body mass and the volume of distribution. This gender differencecontributed the inter-animal variability for BHL011 pharmacokinetics,possibly due to dose-dependent non-linearity resulting from C5-mediatedclearance (FIGS. 9A and 9B). Generally the inter-animal variability waslow for BHL006 (FIGS. 10A and 10B) and BHL009 (FIGS. 11A and 11B) withthe exception of one female in the BHL006 dose cohort (2939) whichdisplayed accelerated clearance. The reasons for accelerated clearancein animal 2939 are unknown.

In the presence of constitutive synthesis of C5 and hFcRn, the highaffinity IgG2/4 anti-05 antibody (BHL011) had a mean terminal half-lifeof 6 days and was cleared from circulation by ˜98% at 21 days (FIGS. 12and 13; Table 8). The mean clearance rate for a pH-dependent anti-C5antibody with an IgG2/4 Fc region (BHL006) was attenuated, with a meanbeta-phase half-life of 16-19 days. An additional ˜2-fold increase inhalf-life was observed for a pH-dependent anti-C5 antibody with anIgG2/4 Fc region with improved affinity for hFcRn (BHL009 half-life ˜36days). These parameters are consistent with those observed for IgG2/4antibodies with and without M428L, N434S substitutions in the absence ofantigen in hFcRn mice. These results demonstrate that pH-dependent C5binding and increased affinity for FcRn confer additive effects toextend the PK exposure of anti-C5 antibodies.

TABLE 8 Animal Body Weight C_(MAX) Half-life Antibody Designation Gender(g) (μg/mL) (days) BHL011 2929 M 37.8 519.8 7.2 2930 M 33.5 512.2 7.12963 F 23.2 805.0 6.2 2964 F 20.2 814.6 5.0 2965 F 23.4 823.5 4.4 Mean =6.0 BHL006 2905 M 37.5 361.6 15.4 2906 M 36.1 378.8 19.1 2939 F 21.8836.0 4.6 2940 F 23.9 635.3 21.6 2941 F 20.0 906.9 20.1 Mean = 16.2BHL009 2913 M 31.2 402.6 45.8 2914 M 31.0 606.7 45.0 2947 F 21.3 724.933.2 2948 F 22.3 590.1 22.8 2949 F 20.9 652.8 33.1 Mean = 36.0

Pharmacodynamics of Anti-Mouse C5 Antibodies in Human FcRn TransgenicMice

The pharmacologic activity of the anti-mouse C5 antibodies in serumsamples was evaluated ex vivo in a complement classical pathway-mediatedchicken erythrocyte (chicken red blood cells; cRBC) hemolysis assay.Hemolytic activity was calculated as a percentage of the activity inpre-dose samples and are shown in FIGS. 14-16. Males are represented assolid lines and females as dashed lines. Antagonism of ex vivo hemolyticactivity is proportional to the concentration of total antibody in thesample. The gender difference in the duration of antagonism of hemolyticactivity was pronounced for BHL011 (FIG. 14) corresponding to the bodymass-dependent inter-animal variability for BHL011 PK (FIG. 9).Generally the inter-animal variability was low for BHL006 (FIG. 15) andBHL 009 (FIG. 16) with the exception of the female in the BHL006 dosecohort (2939) which displayed accelerated antibody clearance (FIG. 10).

Differences in the correlation between total antibody serumconcentration and antagonism of ex vivo hemolytic activity areproportional to the affinity of the antibody for C5. The high affinityantibody (BHL011) nearly completely suppressed hemolytic activity at˜200 μg/mL (FIG. 17) while the weaker affinity, pH-dependent anti-C5antibodies require 2 to 3-fold higher concentrations to achieve fullantagonism ex vivo (FIGS. 18 and 19).

Despite this loss in potency in the pH-dependent anti-C5 antibodies,mean activity levels for cRBC hemolysis across animals from each cohortsuggest that they could support an extended dosing interval. At day 14the high affinity anti-C5 (BHL011) treated animals had mean hemolyticactivity levels of >40%, while the pH-dependent anti-C5 (BHL006 andBHL009) treated animals maintained mean hemolytic activity levels <40%through day 21 and 28, respectively (FIG. 20).

The significant extension in the half-life and corresponding duration ofantagonism of the antibodies with pH-dependent binding to mouse C5(BHL006 and BHL009) relative to the high affinity anti-mouse C5 antibody(BHL011) was consistent with studies described in Examples 4 and 7 inwhich a pH-dependent anti-human C5 antibody (BNJ421, BNJ423 or BNJ441)exhibited a similar increase half-life relative to its high affinitycounterpart (EHL000 or eculizumab) in mice co-administered human C5.These findings further substantiate the notion that engineeringpH-dependent antigen binding through select histidine substitutions inthe CDRs can significantly attenuate antigen-mediated clearance thoughC5, enabling the free antibody to be recycled back to the circulation.Furthermore, the combination of pH-dependent antigen binding andenhanced affinity for FcRn in BHL009 was additive in the effects on PKproperties, doubling the half-life over pH-dependent binding alone(BHL006). These observations are consistent with the hypothesis thatpH-dependent binding to C5 in combination with improved affinity forFcRn may provide a significant extension in the PK parameters andduration of therapeutic PD observed for eculizumab to enable ≧monthlydosing.

Example 7. Generation of a Variant Eculizumab with pH-Dependent Bindingto C5 and Enhanced FcRn-Mediated Recycling

An antibody was generated using eculizumab as a parent molecule.Relative to eculizumab, the variant antibody (designated BNJ441)contained four amino acid substitutions in the heavy chain, Tyr-27-His,Ser-57-His, Met-429-Leu and Asn-435-Ser (note that positions 429 and 435of BNJ441 correspond to positions 428 and 434 under the EU numberingsystem). The amino acid sequence for the heavy chain polypeptide isdepicted in SEQ ID NO:14. The amino acid sequence for the light chainpolypeptide is depicted in SEQ ID NO:11. These mutations were engineeredto enable an extended dosing interval with BNJ441 (cf. eculizumab) byincreasing the circulating half-life through two distinct mechanisms:(1) reducing antibody clearance through target-mediated antibodyclearance and (2) increasing the efficiency of FcRn-mediated antibodyrecycling.

The two amino acid substitutions in the first and second complementaritydetermining regions (CDRs) of the heavy chain variable region,Tyr-27-His and Ser-57-His, weaken the affinity dissociation constant(K_(D)) of BNJ441 for C5 by ˜17-fold at pH 7.4 and ˜36-fold at pH 6.0compared with eculizumab. The two mutations in the third heavy chainconstant region domain (CH3), Met-429-Leu and Asn-435-Ser, increase theaffinity of BNJ441 for FcRn by 10-fold at pH 6.0 compared to eculizumab.

Binding Kinetics (Antibodies to C5)

The kinetics of BNJ441 or eculizumab binding to C5 were determined viasurface plasmon resonance (SPR) on a BIAcore 3000 instrument using ananti-Fc capture method at pH 8.0, 7.4, 7.0, 6.5 and 6.0. Goat anti-humanIgG (Fc) polyclonal antibody (KPL #01-10-20) was diluted to 0.1 mg/mL in10 mM sodium acetate pH 5.0 and immobilized on two flow cells of a CM5chip for 8 min by amine coupling. The test antibody (BNJ441 oreculizumab) was diluted to 0.20 μg/mL in running buffer (HBS-EP; 0.01 MHEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE LifeSciences, catalogue number: BR1001-88). The diluted antibody was theninjected on one flow cell (20 μL for pH 7.4 experiment and 40 μL for pH6.0 experiment) followed by injections of varying concentrations of C5on both cells. The running buffer was titrated with 3M HCl for pH 7.0,6.5 and 6.0 kinetics and with 0.5M NaOH for the pH 8.0 kinetics. Thesurface was regenerated each cycle with 20 mM HCl, 0.01% P20. The datawere processed with a 1:1 Langmuir model using BIAevaluation 4.1software (BIAcore AB, Uppsala, Sweden) with ‘double referencing’.

The dissociation rates of C5 from BNJ441 or eculizumab at pH 8.0, 7.4,7.0, 6.5 and 6.0 were determined via SPR on a BIAcore 3000 instrumentusing the anti-Fc capture method described above with the followingmodifications. The diluted test antibodies were injected on one flowcell followed by an injection of 6 nM C5 on both cells. Immediatelyfollowing the C5 injection, 250 μL of running buffer at various pH'swere injected. Running buffers were prepared as described above. Thedata were processed using BIAevaluation 4.1 software (BIAcore AB,Uppsala, Sweden) with ‘double referencing’. The % dissociation of C5from BNJ441 and eculizumab was calculated by taking the difference indissociation at t=0 and t=300 seconds.

Binding Kinetics (Antibodies to FcRn)

The kinetics of BNJ441 or eculizumab binding to human FcRn weredetermined via SPR on a BIAcore 3000 instrument using an F(ab′)2 capturemethod at pH 7.4, and 6.0. Goat F(ab′)2 anti-human IgG F(ab′)2 (RocklandImmunochemicals, Catalogue number: 709-1118) diluted to 0.04 mg/mL in 10mM sodium acetate pH 5.0, was immobilized on two flow cells of a CM5chip for 7 minutes by amine coupling. The test antibody (BNJ441 oreculizumab) was diluted to 2 μg/mL in running buffer ((HBS-EP; 0.01 MHEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20; GE LifeSciences, Cat. # BR1001-88). The diluted antibody was then injected onone flow cell followed by injections of FcRn on both cells. The runningbuffer was titrated with 3M HCl for pH 6.0 kinetics. The surface wasregenerated each cycle with 10 mM Glycine HCl, pH 1.5). The data wereprocessed with a 1:1 Langmuir model using BIAevaluation 4.1 software(BIAcore AB, Uppsala, Sweden) with ‘double referencing’.

Results of Binding Studies

The kinetics of antibody:C5 binding were found to be pH-dependent witheffects on both association and dissociation rates are shown in Table 9.

TABLE 9 Association Rate: Dissociation Rate: Dissociation Constant: pHK_(a) (1/M*s) K_(d) (1/s) K_(D) (M) Chi² BNJ441 8.0 6.25 * 10⁵ 1.33 *10⁻⁴ 2.13 * 10⁻¹⁰ 0.055 7.4 4.62 * 10⁵ 2.27 * 10⁻⁴ 4.91 * 10⁻¹⁰ 0.0457.0 4.28 * 10⁵ 3.90 * 10⁻⁴ 9.11 * 10⁻¹⁰ 0.028 6.5 4.08 * 10⁵ 8.94 * 10⁻⁴2.19 * 10⁻⁹  0.172 6.0 1.63 * 10⁵ 3.54 * 10⁻³ 2.18 * 10⁻⁸  0.373Eculizumab 8.0 1.39 * 10⁶ 2.04 * 10⁻⁵ 1.47 * 10⁻¹¹ 0.104 7.4 1.10 * 10⁶3.23 * 10⁻⁵ 2.93 * 10⁻¹¹ 0.094 7.0 8.86 * 10⁵ 6.34 * 10⁻⁵ 7.15 * 10⁻¹¹0.032 6.5 8.41 * 10⁵ 1.73 * 10⁻⁴ 2.06 * 10⁻¹⁰ 0.037 6.0 7.05 * 10⁵4.28 * 10⁻⁴ 6.06 * 10⁻¹⁰ 0.092

In an attempt to model the relative rates of dissociation of antibody:C5complexes after pinocytosis and acidification of the early endosome,antibody:C5 complexes were allowed to form in a pH 7.4 buffer, then thebuffer pH conditions were switched during dissociation. The percent ofantibody complex dissociation (estimated by the decrease in resonanceunits [RUs]) after 300 seconds was calculated for each pH condition(Table 10). Only BNJ441 at pH 6.0 resulted in greater than 50%antibody:C5 complex dissociation after 5 minutes.

TABLE 10 RU RU pH 0 sec 300 sec % Dissociation BNJ441 8.0 55.4 53.5 3.47.4 55.7 52.0 6.6 7.0 55.2 49.1 11.0 6.5 55.2 39.4 28.6 6.0 55.8 22.360.0 Eculizumab 8.0 70.2 69.7 0.8 7.4 70.0 69.5 0.7 7.0 71.3 69.9 2.06.5 71.2 67.8 4.7 6.0 71.6 62.9 12.2

FIGS. 21A and 21B depict semi-log and linear plots of the percentage ofdissociation of BNJ441:C5 complexes or eculizumab:C5 complexes as afunction of pH.

The two amino acid substitutions in the first and second complementaritydetermining regions (CDRs) of the heavy chain variable region,Tyr-27-His and Ser-57-His, weaken the affinity dissociation constant(K_(D)) of BNJ441 for C5 by ˜17-fold at pH 7.4 and ˜36-fold at pH 6.0compared with eculizumab. It is unclear if the pH-dependence in theaffinity of BNJ441 for C5 is the result of changes in the protonationstate of the histidines introduced at positions 27 and/or 57, or simplyan overall weakening of the affinity for C5. It has been observed inother anti-C5 antibodies, however, that these mutations in combinationwith additional histidine substitutions, resulted in much morepronounced losses of affinity at pH levels below 6.5. The two mutationsin the third heavy chain constant region domain (CH3), Met-429-Leu andAsn-435-Ser, strengthen the affinity of BNJ441 for FcRn by ˜10-fold atpH 6.0 compared to eculizumab.

PK Properties of the BNJ441 Antibody

The BNJ441 antibody and eculizumab were evaluated in mice that wereimmunodeficient (NOD/scid) and C5 deficient. A single dose of 100 μg ofBNJ441 or eculizumab in 200 μL of phosphate buffered saline (PBS) wasadministered by intravenous (i.v.) injection to each of eight mice.Serum was collected from each of the mice at days one, three, seven, 14,21, 28, and 35 following the administration. The concentration of eachantibody in the serum was measured by ELISA.

As shown in FIG. 22, in the absence of human C5, the serum antibodyconcentrations declined similarly in mice dosed with BNJ441 andeculizumab over a 35 day period. However, in the presence of human C5,eculizumab serum concentrations declined rapidly to undetectable levelsafter day 14 while serum concentration of BNJ441 decayed more slowly andat a consistent rate through duration of study (FIG. 23).

Comparing the PK profiles of the two antibodies in the presence andabsence of human C5, the clearance of eculizumab was accelerated in thepresence of human C5 compared to that in the absence of human C5, whilethe PK profile of BNJ441 in the presence of human C5 was similar to thatof BNJ441 in the absence of human C5 through day 28, and clearance wasonly accelerated between days 28 and 35 (FIG. 24). The half-life ofBNJ441 and the half-life of eculizumab were comparable in the absence ofhuman C5 (25.37±1.02 days for BNJ441 and 27.65±2.28 days foreculizumab). However, in the presence of human C5, BNJ441 demonstratedmore than three-fold increase in half-life in comparison with eculizumab(13.40±2.18 days for BNJ441 vs. 3.93±0.54 days for eculizumab). Itshould be noted that the clearance rate of BNJ441 did not differsignificantly in the presence or absence of human C5 through day 28. SeeTable 11.

TABLE 11 Treatment Group Animal # Half-life (days) BNJ441 2009 26.992011 25.55 2212 24.5 2213 20.34 2214 27.18 2215 24.35 2216 28.65 Mean =25.37 SE = 1.02 Eculizumab 2201 30.65 2202 16.85 2203 27.02 2204 28.542205 19.7 2206 35.47 2207 33.77 2208 29.18 Mean = 27.65 SE = 2.28BNJ441 + Human C5 2225 24.31 2226 13.45 2227 N/A 2228 13.48 2229 16.092230 8.55 2231 11.25 2232 6.66 Mean = 13.40 SE = 2.18 Eculizumab + HumanC5 2217 3.35 2218 2.72 2219 7.45 2220 3.26 2221 2.74 2222 3.93 2223 4.52224 3.51 Mean = 3.93 SE = 0.54

Serum Hemolytic Activity

To determine the effect of the histidine substitutions on hemolyticactivity of the antibody, an ex vivo hemolytic assay was performed asdescribed in Example 6. In the presence of BNJ441, or eculizumab,terminal complement activity was consistent with the respective PKprofiles of each antibody (FIG. 25)—that is, the level of inhibition ofserum hemolytic activity was proportional to the concentration of eachantibody remaining in the serum. Both antibodies conferred near totalinhibition of hemolysis through day 3. However, eculizumab showed noantagonism by day 14, whereas BNJ441 retained about 83% inhibition byday 14 and partial complement inhibition through day 28.

CONCLUSION

The findings from this study suggest that in the presence of human C5,BNJ441 showed more than three-fold extension in half-life compared witheculizumab. In addition, the serum half-life of BNJ441 relative toeculizumab translated into an extended pharmacodynamic profile, asevidenced by prolonged hemolytic inhibition.

Example 8. Safety, Tolerability PK and PD of BNJ441 in Healthy HumanSubjects

The safety, tolerability, PK and PD of BNJ441 was assessed in a Phase 1,randomized, blinded, placebo-controlled, single ascending dose (SAD)human clinical study, wherein BNJ441 was administered intravenously tohealthy subjects.

BNJ441 was formulated in a sterile, preservative-free, aqueous solutionwith formulation excipients. The BNJ441 formulation did not contain anyunusual excipients, or excipients of animal or human origin. Theformulation was phosphate-buffered to a pH of 7.0. The componentsincluded BNJ441 10 mg/ml, sodium phosphate monobasic 3.34 mM, sodiumphosphate dibasic 6.63 mM, sodium chloride 150 mM, polysorbate 80 0.02%and Q.S. water.

The BNJ441 formulation was supplied as a 10 mg/mL antibody solution in a20 mL single-use vial, and was designed for infusion by diluting it intocommercially available saline (0.9% sodium chloride injection, Ph Eur)for IV administration.

TABLE 12 Phase 1 Clinical Trial in Healthy Volunteers Protocol Popu-Dosing Number Title Study Design lation Regimen BNJ441- Phase 1,randomized, First-in-human, healthy Cohort 1: HV-101 blinded, placebo-randomized, volun- 200 mg controlled, single placebo- teers BNJ441ascending-dose study controlled, (4 active, to evaluate BNJ441double-blind, 2 placebo) safety, tolerability, single Cohort 2: PK, andPD as a single ascending-dose 400 mg dose administered BNJ441 IV tohealthy (6 active, subjects 2 placebo)

Ten healthy subjects received a single dose of BNJ441. Four subjectsreceived a dose of 200 mg and six subjects received a dose of 400 mg.The PK and safety data for this study were determined and discussedbelow.

Pharmacokinetics

Serum BNJ441 concentration-time profiles following IV administration of200 mg and 400 mg doses are depicted in FIG. 26. Concentration-time datawere available for up to Day 90 (2136 hours) and Day 57 (1344 hours),following 200 mg and 400 mg doses, respectively. Mean serumconcentrations remained above 50 μg/mL for 2 to 4 days (48 to 96 hours)after the 200 mg dose, and 14 to 21 days (336 to 504 hours) after the400 mg dose.

A summary of BNJ441 PK parameters is reported in Table 12 below. Thegeometric mean (CV) C_(max) of BNJ441 was 78.5 (10.2%) μg/mL followingthe 200 mg dose, and 139 (16.2%) μg/mL following the 400 mg dose. Theobserved median (range) t_(max) was 2.4 (0.79 to 8.0) hours for the 200mg dose, and 0.58 (0.58 to 1.1) hours for the 400 mg dose after thestart of infusion. Geometric mean (CV) AUC_((0-56 days)) is 32,800(8.6%) μ-hr/mL for the 200 mg dose, and 58,100 (18.9%) μg-hr/mL for the400 mg dose. Geometric mean C_(max) and AUC_((0-56 days)) indicate thatexposure increased in an apparent dose-proportional manner. Thegeometric mean t_(1/2) (CV) is 38.5 (18.4%) days, and 32.9 (13.3%) daysfor the 200 mg and 400 mg doses, respectively.

In summary, the PK data suggest mean BNJ441 C_(max) andAUC_((0-56 days)) increased in a dose proportional manner, and support amean (standard deviation [SD]) t_(1/2) of 35.5±6.1 days following IVadministration. Analysis of chicken red blood cell (cRBC) hemolysis dataindicate terminal complement was completely inhibited for up to 2 daysafter a single 400 mg IV dose, when BNJ441 concentrations were greaterthan 100 μg/mL.

TABLE 12 Summary of Pharmacokinetic Parameters for BNJ441 Following IVAdministration of 200 mg or 400 mg to Healthy Volunteers DoseDescriptive C_(max) C_(max)/Dose t_(max) AUCτ^(a) AUCτ/Dose t_(1/2) (mg)Statistic (ug/mL) (ug/mL/mg) (h) (h*ug/mL) ((h*ug/mL)/mg) (day) 200 N 44 4 4 4 4 Geometric Mean 78.5 0.392 2.40^(b) 32,800 164 38.5 CV %Geometric 10.2 10.2 0.79-8.0^(c) 8.6 8.6 18.4 Mean 400 N 6 6 6 6 6 6Geometric Mean 139 0.348 0.58^(b) 58,100 145 32.9 CV % Geometric 16.216.2 0.58-1.1^(c) 18.9 18.9 13.3 Mean ^(a)AUCτ = AUC_((0-56 days))^(b)median ^(c)range

Pharmacodynamics

The ability of BNJ441 to inhibit cRBC hemolysis over time was alsoassessed, as illustrated in FIG. 27. Mean cRBC hemolysis activity wasrelatively stable in subjects who received placebo. The onset of cRBChemolysis inhibition was rapid, with complete terminal complementinhibition observed at the end of infusion (0.29 hours for the 200 mgdose, and 0.58 hours for the 400 mg dose). BNJ441 had a dose-dependentduration of action, which lasted for 4 to 14 days.

The relationship between BNJ441 concentration and cRBC hemolysis wereplotted and are depicted in FIG. 28. As shown in Figured 28, completeterminal complement inhibition occurred at BNJ441 concentrations above50 μg/mL, with no inhibition was observed at BNJ441 concentrations below25 μg/mL.

Example 9. Single Dose Study in Cynomologous Monkeys

A single IV dose of BNJ441 was administered to cynomolgus monkeys atdoses of 60 or 150 mg/kg (n=4 for each dose group; 2 males and 2 femalesper dose group) as a 2-hour infusion. Blood samples for BNJ441 analysiswere collected from Day 1 to Day 112.

All BNJ441-treated monkeys were screened for the presence ofCynomologous anti-human antibodies (CAHA) before dosing (0 hour), and onDays 8, 14, 28, 56, 84, and 112.

All monkeys in the 60 and 150 mg/kg dose group were confirmed positiveon at least a single occasion, except Animal 2002 in the 150 mg/kg dosegroup. The presence of CAHA in Animal 2002, or at non-positive timepoints for the other animals, cannot be excluded, due to possibleinterference of the administered BNJ441 with the biotinylated-BNJ441 andruthenylated-BNJ441 bridging assay. The positive CAHA results wereobserved in the 60 mg/kg dose group from Day 56 to 112 after dosing, andin the 150 mg/kg dose group from Day 28 to 112 after dosing. The firstconfirmed CAHA-positive sample in the 60 mg/kg was on Day 56 (Animals1002 and 1503), 2 on Day 84 (Animals 1002 and 1503), and 3 on Day 112(Animals 1001, 1002, and 1502). Animal 1503, who was CAHA positive onDays 56 and 84, was no longer CAHA-positive on Day 112. The firstconfirmed CAHA-positive sample in the 150 mg/kg dose group was Animal2502 on Day 28, followed by 2 monkeys on Day 56 (Animals 2001 and 2502),3 monkeys on Day 84 (Animals 2001, 2501, and 2502), and 3 monkeys on Day112 (Animals 2001, 2501 and 2502).

Individual BNJ441 concentration-time profiles were calculated. In the 60mg/kg dose group, all monkeys had quantifiable plasma BNJ441concentrations through the Day 112 PK sample, whereas in the 150 mg/kgdose group, only 1 monkey (Animal 2002) had quantifiable plasma BNJ441concentrations through Day 112. Concentration-time data indicated aprolonged residence of BNJ441 in the systemic circulation of monkeys.

Noncompartmental PK parameters and summary statistics for BNJ441 werecalculated for all monkeys by dose level, and shown in Tables 13 and 14for the 60 mg/kg and 150 mg/kg dose levels, respectively. Consistentwith duration of infusion, median t_(max) was 2 hours for the 60 mg/kgand 150 mg/kg dose levels. One monkey in the 150 mg/kg dose group,Animal 2501, had a t_(max) of 12 hours after dosing, and had arelatively flat profile from 2 to 12 hours after dosing, with the12-hour post dose sample concentration approximately 5% greater thanthat observed at 2 hours after dosing. Geometric mean C_(max), AUC_(∞),and AUC_(last) all increased with increasing dose. Geometric meandose-normalized C_(max) values were similar across the 2 doses,indicating a dose-proportional increase in peak BNJ441 concentrationwith an increase in dose, but geometric mean dose-normalized AUC_(∞)values were different between the dose groups. This difference is likelydue to CAHA-mediated increase in BNJ441 CL in the 150 mg/kg dose group;clearance of BNJ441 was approximately 37% greater in monkeys dosed with150 mg/kg compared to the monkeys dosed with 60 mg/kg. Geometric meanV_(ss) was similar (within 12%) between the 2 dose groups.

TABLE 13 Summary of Noncompartmental Pharmacokinetic Parameters ofBNJ441 (60 mg/kg Dose) Dose C_(max) C_(max)/ t_(max) AUC_(last) AUC_(∞)AUC_(∞)/ V_(ss) CL t_(1/2) t_(1/2) Animal (mg/kg) (mg/mL) Dose¹⁾ (hr)(hr × mg/mL) (hr × mg/mL) Dose²⁾ (mL/kg) (mL/h/kg) (hr) (day) 1001 601.92 0.0320 2.0 546 555 9.25 63.4 0.108 479 20.0 1002 60 1.90 0.0317 2.0470 475 7.92 55.3 0.126 474 19.8 1502 60 1.45 0.0242 2.0 598 614 10.264.9 0.0977 547 22.8 1503 60 1.44 0.0240 2.0 701 745 12.4 73.7 0.0806649 27.1 N 4 4 4 4 4 4 4 4 4 4 Mean 1.68 0.0280 2.00 579 597 9.95 64.30.103 537 22.4 SD 0.269 0.00448 NA 97.0 113 1.89 7.53 0.0191 81.7 3.40Min 1.44 0.0240 2.00 470 475 7.92 55.3 0.0806 474 19.8 Median 1.680.0279 2.00 572 585 9.74 64.2 0.103 513 21.4 Max 1.92 0.0320 2.00 701745 12.4 73.7 0.126 649 27.1 CV % 16.0 16.0 NA 16.8 19.0 19.0 11.7 18.515.2 15.2 Geometric Mean 1.66 0.0277 NA 573 589 9.82 64.0 0.102 533 22.2CV % Geometric Mean 16.2 16.2 NA 16.9 19.0 19.0 11.8 19.0 14.8 14.8¹⁾Units are mg/mL/mg/kg ²⁾Units are h × mg/mL/mg/kg hr = hour; NA = notapplicable;

TABLE 14 Summary of Noncompartmental Pharmacokinetic Parameters ofBNJ441 (150 mg/kg Dose) Dose C_(max) C_(max)/ t_(max) AUC_(last) AUC_(∞)AUC_(∞)/ V_(ss) CL t_(1/2) t_(1/2) Animal (mg/kg) (mg/mL) Dose¹⁾ (hr)(hr × mg/mL) (hr × mg/mL) Dose²⁾ (mL/kg) (mL/h/kg) (hr) (day) 2001 1503.79 0.0253 2.0 787 787 5.25 52.6 0.191 61.0 2.54 2002 150 4.51 0.03012.0 1160 1220 8.15 89.8 0.123 759 31.6 2501 150 4.48 0.0299 12.0 14601460 9.71 58.8 0.103 87.6 3.65 2502 150 4.40 0.0293 2.0 1030 1030 6.8637.5 0.146 54.1 2.25 N 4 4 4 4 4 4 4 4 4 4 Mean 4.30 0.0286 4.50 11101120 7.49 59.7 0.141 240 10.0 SD 0.340 0.00227 NA 279 285 1.90 22.00.0377 346 14.4 Min 3.79 0.0253 2.00 787 787 5.25 37.5 0.103 54.1 2.25Median 4.44 0.0296 2.00 1100 1130 7.50 55.7 0.134 74.3 3.09 Max 4.510.0301 12.0 1460 1460 9.71 89.8 0.191 759 31.6 CV % 7.91 7.91 NA 25.225.3 25.3 36.9 26.8 144 144 Geometric Mean 4.28 0.0286 NA 1080 1100 7.3056.8 0.137 122 5.07 CV % Geometric Mean 8.25 8.25 NA 26.1 26.7 26.7 37.226.7 190 190 ¹⁾Units are mg/mL/mg/kg ²⁾Units are h × mg/mL/mg/kg hr =hour; NA = not applicable

Example 10. A Comparative Assessment of BNJ441, Eculizumab and h5G1.1Binding to Fc-Gamma Receptors C1q In Vitro

The binding of three humanized antibodies, BNJ441, eculizumab andh5G1.1-IgG1 to molecules known to be mediators of antibody effectorfunction was examined. BNJ441, eculizumab, and h5G1.1-IgG1 each haveunique functional and therapeutic profiles. However, all three arehumanized antibody antagonists of terminal complement, which bind a verysimilar epitope on human complement component C5 and prevent itscleavage during complement activation into its active metabolites, C5aand C5b.

BNJ441, eculizumab, and h5G1.1-IgG1 are identical in their light chainsequences, each having a humanized variable region and human IgKappaconstant region. BNJ441 and eculizumab both contain a human hybridIgG2-G4 Fc, which includes the CH1 region, hinge and first 29 aminoacids of the CH2 region from human IgG2 fused to the remainder of theCH2 and CH3 regions of human IgG4. This chimeric Fc combines the stabledisulfide bond pairing of an IgG2 with the effector less properties ofan IgG4. Since BNJ441 and eculizumab are directed against a solubleantigen, it was not possible to directly assess their capacity toinitiate antibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC). Instead, direct measurements ofBNJ441 or eculizumab binding to Fc gamma receptors (FcγRs) andcomplement component C1q were performed and it was inferred that in theabsence of binding they cannot mediate ADCC or CDC, respectively.h5G1.1-IgG1 (an IgG1 isotype antibody with the same humanized variableregion as eculizumab) was included as a control. The IgG1 isotype Fcregion is expected to bind effector function molecules fully, thoughh5G1.1-IgG1 itself would not elicit ADCC or CDC in the absence of a cellassociated antigen.

As discussed above in Example 7, BNJ441 was engineered from eculizumabto increase its half life in vivo by introducing 4 amino acidssubstitutions in the heavy chain. Two amino acid changes in thehumanized heavy chain variable region, Tyr-27-His and Ser-57-Hisrespectively (heavy chain amino acid numbering according to Kabat etal.), were introduced to destabilize binding to C5 at pH 6.0 withminimal impact on binding to C5 at pH 7.4. Mutations in the third heavychain constant region domain (CH3), Met-428-Leu and Asn-434-Ser, wereintroduced to enhance binding to the human neonatal Fc receptor (FcRn).Taken together these mutations were designed to significantly attenuateantigen-mediated drug clearance by increasing dissociation ofantibody:C5 complexes to free antibody in the acidified environment ofthe early endosome after pinocytosis, and to increase the fraction ofantibody recycled from the early endosome back into the vascularcompartment by FcRn.

In these studies, multimeric interactions of the FcγR subclasses (FcγR1,FcγRIIa, FcγRIIb, FcγRIIb/c, FcγRIIIa and FcγRIIIb) with all threeantibodies were evaluated in an enzyme linked immunosorbent assay(ELISA) and monomeric interactions with FcγRs were evaluated usingsurface plasmon resonance (SPR). Biolayer interferometry and SPR wasused to examine the binding of C1q to the three antibodies. The reagentsused to conduct these analyses are shown in Table 15.

TABLE 15 Antibodies and Protein Reagents Reagent Source ConcentrationBNJ441 Alexion 10 mg/mL Eculizumab Alexion 10 mg/mL Goat anti-humanJackson 1.1 mg/mL F(ab′)2-biotin Immunolabs HRP-streptavidin Invitrogen1.25 mg/mL h5G1.1-IgG1 Alexion 1.43 mg/mL 8.11 mg/mL C1q Complement 1mg/mL Technology Human FcγRI R&D systems 100 μg/mL (CD64) Human FcγRIIaR&D systems 100 μg/mL (CD32a) Human FcγRIIb/c R&D systems 100 μg/mL(CD32b/c) Human FcγRIIIa R&D systems 100 μg/mL (CD16a) Human FcγRIIIbR&D systems 100 μg/mL (CD16b)

Binding of Multivalent Antibody Complexes to FcγRs

Antibody complexes were prepared by incubating BNJ441, Eculizumab orh5G1.1-hG1 overnight with goat-anti-human F(ab′)2-biotin (JacksonImmunolabs), at a 2:1 antibody: F(ab′)2 molar ratio in phosphatebuffered saline (PBS) in a 1.5 mL microfuge tube.

Microtiter plates pre-coated with Ni-NTA (Qiagen) were incubated with 50μL/well of 6× histidine-tagged human FcγRs (FcγRI, FcγRIIa, FcγRIIb/c,FcγRIIIa or FcγRIIIb), at a receptor concentration of 5 μg/mL in PBS,overnight at 4° C. The plate was then washed 3 times with PBS/0.05%Tween-20. After washing, 50 μL of antibody complexes in PBS/0.05%Tween-20 were incubated in the plate for 60 min at room temperature(RT). After washing the plate with PBS/0.05% Tween-20, 50 μL ofstreptavidin-HRP (Invitrogen) in PBS/0.05% Tween-20 was added to theplate and incubated for 60 min at RT. Following this incubation andwashes, 75 μL of TMB-ELISA substrate (3, 3′, 5, 5′-tetramethylbenzidine,Thermo Scientific) was added. The reaction was stopped with 754, of 2 MH2504, and the absorbance read at 450 nm.

Samples were run in duplicate and data were presented as mean values.Results were entered into a spreadsheet program. The absorbance at 450nm of each concentration of antibody immune complex or in the absence ofantibody immune complexes plotted as a graphical representation. The keydissociation constants were calculated and are summarized in Table 16and discussed below.

Binding of Monovalent Antibodies to FcγRs

The kinetics of BNJ441, eculizumab, and h5G1.1-IgG binding to FcγRs weredetermined via SPR on a BIAcore 3000 instrument using directimmobilization. BNJ441, eculizumab, and h5G1.1 were diluted in 10 mMsodium acetate pH 5.0, was immobilized on one flow cell of a CM5 chip byamine coupling. A second flow cell was used as a reference surface.Concentrations of FcγRs diluted in running buffer (HBS-EP, pH 7.4) wereinjected on both cells. The surface was regenerated each cycle with 20mM HCl, 0.01% P20. The data was analyzed using a steady state affinitymodel in BIAevaluation 4.1 software (BIAcore AB, Uppsala, Sweden) with‘double referencing’.

The kinetics of h5G1.1-IgG1 binding to FcγRI was assessed via singlecycle kinetics due to its stronger affinity. The antibody was diluted in10 mM sodium acetate at pH 5.0 and directly immobilized on one flow cellof a CM5 chip by amine coupling. A second flow cell was used as areference surface. Concentrations of FcγR1 diluted in running buffer(HBS-EP, pH 7.4) were injected on both cells. This assay required noregeneration. The data was analyzed using a titration kinetics 1:1 modelin BIAevaluation 4.1(Biacore AB, Uppsala, Sweden) software with ‘doublereferencing’.

TABLE 16 Dissociation constants for BNJ441, eculizumab and h5G1.1-IgG1binding to monomeric FcyRs BNJ441, Eculizumab, h5G1.1-IgG1, FcγR K_(D)[μM] K_(D) [μM] K_(D) [μM] RI 3.75 3.78 0.123 RIIa 2.31 2.58 0.8 RIIb/c8.09 9.84 3.06 RIIIa 7.23 6.78 0.85 RIIIb 3.33 3.49 1.89

ELISA assays to detect avidity-driven multimeric interactions ofantibody immune complexes and FcγRs were performed. The results aresummarized in Table 16. BNJ441 and eculizumab displayed no detectablebinding to FcγRI, FcγRIIb/c, FcγRIIIa or FcγRIIIb and a 4-fold to 8-foldweaker association with FcγRIIa, respectively. Dissociation constants(K_(D)) for monomeric FcγR binding to BNJ441 and eculizumab derived bySPR confirmed that FcγR interactions are very weak and nearlyindistinguishable between the two antibodies: FcγRI (˜4 μM), FcγRIIa (˜2μM), FcγRIIb (˜9 μM), FcγRIIIa (˜7 μM) and FcγRIIIb (˜3 μM).Dissociation constants for the IgG1 isotype control (h5G1.1-IgG1) wereconsistent with high affinity interactions with FcγR1 (123 pM) andmodest increases in binding to the low affinity FcγRs relative to theIgG2-G4 isotype antibodies: FcγRIIa (˜1 μM), FcγRIIb (˜3 μM), FcγRIIIa(˜1 μM) and FcγRIIIb (˜2 μM). See Table 16. No interactions between C1qand BNJ441 or eculizumab were detectable via biolayer interferometry.These results are consistent with the idea that the chimeric humanIgG2-G4 Fc of eculizumab has little to no capacity to elicit effectorfunction through FcγRs or C1q to mediate ADCC or CDC, respectively.Furthermore, these results show that the heavy chain amino acidsubstitutions incorporated in BNJ441 do not significantly alter bindingto these, relative to eculizumab.

Example 11: Tissue Cross Reactive Studies

1. GLP Human Cross-Reactivity Studies

Potential cross reactivity with human tissues was determined usingfluoresceinated BNJ441 (designated BNJ441-FITC) and a control antibody(OX-90G2G4-FITC) with a different antigenic specificity.

BNJ441-FITC produced moderate to intense staining of the positivecontrol material (purified human complement protein C5 ultraviolet[UV]-resin spot slides, designated hC5) but did not specifically reactwith the negative control material (human hypercalcemia of malignancypeptide, amino acid residues 1-34, UV-resin spot slides, designatedPTHrP 1-34). The control article, OX-90G2G4-FITC, did not specificallyreact with either the positive or negative control materials. Theexcellent specific reactions of BNJ441-FITC with the positive controlmaterial and the lack of specific reactivity with the negative controlmaterial, as well as the lack of reactivity of the control article,indicated that the assay was sensitive, specific, and reproducible.Staining with BNJ441-FITC was observed in the human tissue panel, assummarized below:

-   -   Proteinaceous material in most human tissues    -   Cytoplasm and/or cytoplasmic granules in the following tissue        elements:        -   mononuclear cells in the colon, esophagus, lymph node,            parathyroid, spleen, and tonsil        -   platelets in blood smears and bone marrow        -   megakaryocytes in the bone marrow        -   epithelium in the fallopian tube, liver (hepatocytes),            pancreatic ducts, and cervix        -   mesothelium in the lung

Because C5 is a circulating serum protein, the staining of proteinaceousmaterial was expected. Mononuclear cells such as monocytes, macrophages,and dendritic cells, as well as platelets, have been reported to secreteC5; therefore, the staining of these cell types with BNJ441-FITC wasalso expected. Additionally, mesothelial cell lines have been shown toproduce C5. However, no literature was available describing theexpression of C5 by the epithelial cell types stained with BNJ441-FITCin the current study, or megakaryocytes, although platelets, which havebeen shown to produce C5, are derived from megakaryocytes. Therefore,staining of epithelial cell types might represent either previouslyunrecognized sites of C5 expression, or tissue cross-reactivity with aprotein sequence or structure from a similar but unrelated protein orother constituent(s) of the tissue sections. However, with the exceptionof staining of proteinaceous material, all staining observed in thisstudy was cytoplasmic in nature, and it is unlikely that the cytoplasmand cytoplasmic structures would be accessible to the test article invivo. In summary, no specific cross-reactivity of BNJ441-FITC stainingwas observed that would lead to the expectation of treatment-relatedtoxicity.

2. GLP Cynomolgus Monkey Tissue Cross-Reactivity Studies

A standard GLP tissue cross-reactivity study was also done using a panelof cynomolgus monkey tissues to examine both off-target and on-targetbinding, with the same reagents used in the human tissue bindingstudies.

Some staining with BNJ441-FITC was observed in the cynomolgus monkeytissue panel, as summarized below:

-   -   Proteinaceous material in most cynomolgus monkey tissues    -   Cytoplasm and/or cytoplasmic granules in the following tissue        elements:        -   mononuclear cells in the lymph node, spleen, and tonsil        -   epithelium in the fallopian tube

The BNJ441-FITC staining pattern observed in the cynomolgus monkeytissue panel was overall less intense and less frequent than thatobserved in the human tissue panel in the companion human tissuecross-reactivity study. Further, in the human tissue panel, staining ofplatelets, megakaryocytes, pancreatic ductal epithelium, cervicalepithelium, hepatocytes, and mesothelium was observed, although thesetissue elements were not stained in the cynomolgus monkey tissue panel.Moreover, with the exception of staining of proteinaceous material, thestaining observed in this study was cytoplasmic in nature, and it isunlikely that the cytoplasm and cytoplasmic structures would beaccessible to the test article in vivo. Because BNJ441 has been shown tobe exquisitely specific for human C5 (and is not cross-reactive with C5from nonhuman primates), it is likely that the limited binding observedin this study was due to nonspecific binding with an unidentifiedcross-reactive material

Example 12: Potency of BNJ441 Compared to Eculizumab in TerminalComplement Activity Assays

The mutations engineered in BNJ441 to yield pH-dependent binding to C5weaken its affinity at pH 7.4 (approximately 491 pM) by approximately17-fold relative to eculizumab (approximately 29.3 pM) and might beexpected to reduce BNJ441 inhibition potency of C5-mediated terminalcomplement activity compared to eculizumab. To estimate the potencies ofBNJ441 and eculizumab under physiologically relevant conditions,antagonism of complement-mediated hemolysis of red blood cells (RBCs)from 3 commonly used animal models (chicken, sheep, and rabbits) wasassessed in 90% normal human serum.

RBCs and sheep red blood cells (sRBCs) were pre-sensitized withantibodies to initiate activation of the complement classical pathway(CCP). Rabbit red blood cells (rRBCs) were not pre-sensitized and areused as a model of complement alternative pathway (CAP) activation.Antibodies were pre-incubated in serum at 100, 200, and 400 nM to yieldmolar ratios of antigen binding sites to C5 of approximately 0.5:1, 1:1,and 2:1, respectively. Antibody BNJ430 contains the same Fc region asBNJ441, but does not bind human C5, and was included as a negativecontrol. Percent hemolysis was measured at 0, 1, 2, 3, 4, 5, 6, and 8minutes to ensure that reactions were observed under initial velocityconditions.

As shown in FIG. 29, neither BNJ441 nor eculizumab displayed antagonismat 100 nM in cRBC hemolysis. Both antibodies exhibited partialantagonism at 200 nM (approximately 1:1molar ratio of antigen bindingsites to C5), with BNJ441 having reduced potency relative to eculizumab.Inhibition of hemolysis was nearly complete for either antibody whenincubated at a 2:1 molar ratio of antigen binding sites to C5 (400 nM).Results of sRBC hemolysis assays were similar, showing less than 20%hemolysis in the presence of BNJ441 at 200 nM, and near completeinhibition with each antibody at 400 nM (data not shown). TheCAP-mediated rRBC hemolysis assays exhibit higher levels of hemolysis inthe presence of anti-C5 antibodies, with no detectable inhibition at 200nM, and only partial inhibition at 400 nM (data not shown).

In conclusion, the modest loss in potency of BNJ441 relative toeculizumab in these in vitro complement activity assays is consistentwith its weaker affinity for C5. The affinity of BNJ441 for C5 is stillapproximately 1000-fold lower than the concentrations of C5 in vivo andtargeted therapeutic levels of BNJ441, and is therefore unlikely tocompromise its therapeutic efficacy.

Example 13: Selectivity of BNJ441 Compared to Eculizumab in TerminalComplement Activity Assays

To assess the pharmacologic activity of BNJ441 in non-human animalmodels the ability of BNJ441 to antagonize complement-mediated hemolysisof antibody-sensitized cRBCs in serum from chimpanzee, baboon, rhesusmacaque, cynomolgus macaque, beagle, rabbit, guinea pig, rat and mousewere measured. Eculizumab and an anti-mouse-C5 antibody with a humanIgG2/G4 Fc (BNJ430) were used as isotype controls.

Sensitized cRBCs were prepared for each assay from 400 μL of chickenwhole blood in Alsever's (Lampire Biologicals) and washed 4 times with 1mL of GVBS at 4° C. and re-suspended in GVBS at 5×10⁷ cells/mL. Tosensitize chicken erythrocytes, a polyclonal anti-chicken RBC antibody(Rockland) was added to the cells at 150 μg/mL and incubated for 15 minon ice. After washing with GVBS once, the cells were re-suspended inGVBS to a final volume of 3.6 mL.

Complement preserved sera were obtained from Bioreclamation includingserum from the following mammals: human; chimpanzee; baboon; rhesusmacaque; cynomolgous macaque; beagle; rabbit; guinea pig; and rat.Antibodies BNJ441 at 10 mg/ml; eculizumab (10 mg/ml); BNJ430 at 0.873mg/ml were diluted to a final concentration of 0, 60, 300 and 600 nM in30% serum in GVBS and incubated at room temperature for 30 min.Sensitized cRBCs were added to the antibody/serum mixture at 30 μL perwell (2.5×10⁶ cells), incubated at 37° C. for 30 min and reactions werestopped by adding 30 μL of 0.5M EDTA to each well. The plates werecentrifuged at 1800×g for 3 min and 80 μL of the supernatant wastransferred to a new flat-bottom 96-well plate. The absorbance wasmeasured at 415 nm.

As mouse serum is a poor source of classical pathway complementactivity, mouse serum was mixed 1:1 with C5-depleted human serum toassess potential BNJ441 pharmacologic activity in mice. Antibodies werediluted to a final concentration of 0, 60, 300 and 600 nM in 50% totalserum (25% mouse serum, 25% C5 depleted human serum) in GVBS andincubated at room temperature for 30 min. Sensitized cRBCs were added tothe antibody/serum mixture at 30 per well (2.5×10⁶ cells), incubated at37° C. for 30 min and reactions were stopped by adding 30 μL of 0.5MEDTA to each well. The plates were centrifuged at 1800×g for 3 min and80 of the supernatant was transferred to a new flat-bottom 96-wellplate. The absorbance was measured at 415 nm.

Samples containing serum without anti-C5 antibodies with or without 10mM EDTA were used as no lysis or complete lysis controls, respectively.Sample conditions were run in triplicate or duplicate.

Results were entered into a spreadsheet to allow background subtractionof no lysis controls and normalization of percent hemolysis relative tocomplete lysis controls, calculation of mean values (±s.d.) andgraphical representation of the data. Absorbance values for meanbackground from no lysis controls were subtracted from each replicateand sample absorbance was expressed as the percent of lysis in completelysis controls according to the following equation: % of cRBC hemolysisequals (A415 value in each sample replicate sample−mean A415 value in nolysis control)/(mean A415 value in complete lysis control−mean A415value in no lysis control)×100.

The mean and standard deviation of the % cRBC hemolysis for samplereplicates were plotted as a graphical representation (data not shown).

BNJ441 was shown to have no detectable binding to native C5 fromcynomolgus macaque and no pharmacologic activity in vitro in anynon-human sera tested at an 8-fold molar excess of antigen binding sitesto C5. Taken together, these data are consistent with the conclusionthat BNJ441 does not have any relevant pharmacologic activity in anyreadily accessible non-human species suitable for modeling thepharmacokinetics or pharmacodynamics in humans.

Example 14: Physicochemical Characterization of BNJ441

The BNJ441 antibody is a recombinant, humanized antibody, and consistsof two identical 448 amino acid heavy chains and two identical 214 aminoacid light chains. See FIG. 30. The constant regions of BNJ441 includethe human kappa light chain constant region and the hybrid humanIgG2-IgG4 heavy chain constant region (also referred to as “G2/G4”). TheIgG2/G4 constant region was rationally designed to reduce the effectorfunction activation, complement activation, and immunogenicity of theantibody. The heavy chain CH1 domain, hinge region and the first 5 aminoacids of the CH2 domain match human IgG2 amino acid sequence, residues 6to 36 in the CH2 region and common to both human IgG2 and IgG4 aminoacid sequence, while the remainder of the CH2 domain and the CH3 domainmatch human IgG4 amino acid sequence. The heavy and light chain variableregions which form the human C5 binding site consist of human frameworkregions were grafted to murine complementarity-determining regions. Theinter-chain disulfide bonds in the BNJ441 antibody are depicted in FIG.31. The residue numbers are shown in FIG. 31 for all the disulfide bondpairing and N-linked glycan sites.

Table 17 lists the general properties of the BNJ441 antibody. Thetheoretical chemical formula and theoretical average molecular weightfor the main component presented below assume that the antibody containseighteen disulfide bonds, two heavy chain N-terminal pyroglutamations,the clipping of two heavy chain C terminal lysines, and the addition oftwo G0F glycan residues. The number of amino acid residues in BNJ441 hasbeen predicted by amino acid analysis.

TABLE 17 General Properties of the BNJ441 Antibody Property ValueTheoretical Chemical Formula C₆₅₄₂ H₁₀₀₇₂N₁₇₀₄ O₂₁₀₆ S₄₈ TheoreticalAverage Molecular Weight 147,827.62 Da Number of Amino Acids 1324

A stable Chinese hamster ovary (CHO) cell line expressing BNJ441 wasdeveloped for the manufacture of BNJ441. The source CHOK1SV cells usedto generate this cell line were obtained from Lonza Biologics CHOK1SVmaster cell bank 269-M. This cell source was verified to be free ofbacterial and fungal contaminants and all detectable viruses other thancell endogenous retroviral particles that are not infectious. HostCHOK1SV cells were transfected with plasmid pBNJ441.1 and stable cloneswere selected with the MSX. Primary clone 3A5 was selected as theproduction cell line for the manufacture of BNJ441.

Engineering and GMP batches of BNJ441 bulk drug substance batches wereprepared and physicochemically characterized by the tests listed inTable 18. The engineering batch was produced in a pilot plant using CHOcells grown a 200 L bioreactor and the purified material was used in thePK study. The GMP batch was produced using CHO cells grown in the pilotplant using a 200 L bioreactor. The BNJ441 engineering and GMP bulk drugsubstance batches were formulated and tested at approximately 10 mg/mL.The physicochemical properties for the batches are summarized in Table19.

TABLE 18 BNJ441 Physicochemical characterization Test Category TestPurity Analytical Ultracentrifugation Size Intact Molecular WeightAnalysis (MALDI-ToF-MS) Size Intact Molecular Weight Analysis(ESI-ToF-MS) Identity N-Terminal Sequencing Primary structure Amino AcidAnalysis Higher order structure Circular Dichroism SpectrometryGlycosylation pattern N-Linked Oligosaccharides Mass Profiling(MALDI-ToF-MS) Glycosylation pattern Oligosaccharides Glycosylationpattern Monosaccharides Glycosylation pattern Sialic AcidThermostability Differential Scanning Calorimetry Kinetics and SelfBiacore Kinetics and Self-Association Association

TABLE 19 BNJ441 Physicochemical Summary Engineering Batch GMP TestBNJ441 BNJ441 Analytical 99.3% 99.0% Ultracentrifugation % monomerMolecular Weight 148,484 148,522 Analysis MALDI-ToF- MS (Da) MolecularWeight Major isoform Major isoform Analysis ESI-ToF-MS 147830.80147830.72 (Da) Range 147,000- Range 147,000- 149,500 149,500 N-TerminalSequencing PyroQ PyroQ Heavy Chain V V Q Q L L V V Q Q S S G G A A E E VV K K K K P P G G A A S S V V K K V V S S N-Terminal Sequencing D DLight Chain I I Q Q M M T T Q Q S S P P S S S S L L S S A A S S V V G GD D R R V V T T residues per residues per Amino Acid Analysis (#)molecule molecule ASX (106) 105 102 GLX (138) 137 135 SER (166) 170 167GLY (84) 89 88 HIS (22) 26 26 ARG (36) 42 42 THR (110) 106 105 ALA (64)68 67 PRO (88) 93 92 TYR (54) 51 53 VAL (128) 127 129 MET (12) 11 11 ILE(28) 26 27 LEU (94) 92 94 PHE (50) 51 51 LYS (82) 68 73 CircularDichroism Near UV Feature NearUV (nm) NearUV (nm) max 295 295 min 269269 max 266 266 min 262 262 negative deflection 250 250 Far UV FeatureFar UV (nm) FarUV (nm) shoulder 239-231 239-231 max 218 218 min 201 202Deconvolution Decon Decon α-helix 0.030 0.030 3/10 helix 0.026 0.026β-sheet 0.328 0.334 Turns 0.156 0.158 Poly (Pro) II 0.059 0.061Unordered 0.397 0.388 Total¹ 0.996 0.997 Oligosaccharides (MALDI-ToF-MS)m/z (M + Na)+ m/z (M + Na)+ G1F 1647.61 1647.55 G1 1501.52 1501.49 G0F1485.56 1485.51 G0 1339.47 1339.49 G0F-GN 1282.46 1282.39 Man-5 1257.431257.48 Oligosaccharide % % M3N2F 0.00 0.00 G0F-GN 0.66 0.93 G0F 90.4591.26 G1F 8.79 7.7 G2F 0.00 0.00 Man-5 0.09 0.12 aGal1 0.00 0.00 Man-60.00 0.00 aGal2 0.00 0.00 Man-7 0.00 0.00 aGal3 0.00 0.00 SA1-1 0.000.00 SA1-2 0.00 0.00 SA1/aGal4 0.00 0.00 SA1-3 0.00 0.00 SA1-4 0.00 0.00SA2-1 0.00 0.00 SA2-2 0.00 0.00 Total G0F, G1F, G2F 99.24 98.96 Acidic0.00 0.00 High Mannose 0.09 0.12 aGal 0.00 0.00 Neutral 99.99 100.01Monosialylated 0.00 0.00 Disialylated 0.00 0.00 (nmol mono/ (nmol mono/Monosaccharide mg protein) mg protein) GlcNAc 22.14 29.26 GalNAc 0.000.00 Galactose 0.66 0.82 Mannose 20.25 23.24 Fucose 5.38 6.53 Total 4860 % Glycosylation 0.93% 1.16% Sialic Acid (mmol/mol) (mmol/mol) NGNA NDND NANA <LoQ <LoQ Calorimetry T_(m) 67.0° C. 67.0° C. Biacore Kineticsk_(a) (1/MS) 4.44e⁵ 4.86e⁵ K_(d) (1/s) 2.05e⁻⁴ 2.04e⁻⁴ K_(D) (M)4.61e⁻¹⁰ 4.21e⁻¹⁰ Chi² 0.0257 0.0347 Biacore Self-Association K_(D) (M)7.12e⁻³ 2.71e⁻⁴ Chi² 0.147 0.359

Table 19 shows the intact molecular weight determined for theengineering batch was 147830.80 Da and GMP batch was 147830.72 Da. Thevalues were consistent with the calculated major component molecularweight value for BNJ441 of 147,827.62 Da in Table 17, and within the 100ppm mass accuracy of the externally calibrated ESI-ToF-MS. No majorpeaks were observed beyond the 147,000-149,500 Da range. This methodidentified the molecule on the basis of intact molecular weight. Testsamples were injected onto a C4 RP-HPLC column and eluted with anaqueous:organic solvent gradient. The eluate was then electrosprayedinto a ToF mass spectrometer and a spectrum from the upper half of thechromatographic peak was deconvoluted to provide the intact molecularweight.

Table 19 shows the N-Terminal sequence determined for the BNJ441batches. The determined N-Terminal sequences of the heavy chain andlight chain were consistent with the amino acid sequence for BNJ441batches. The heavy chain was found to be blocked with a PyroQ, asexpected, and was de-blocked with pyroglutamate aminopeptidase (PGAP).We determined the primary sequence of the protein at the N-terminus ofthe polypeptide chain by sequential Edman degradation and HPLC analysis.

Table 19 shows the Amino Acid Analysis residues per molecule determinedfor the BNJ441 batches. These values were all consistent with thecalculated number of residues per molecule for BNJ441 based on theprimary sequence, shown in the first column of Table 19. The Amino AcidAnalysis data were acquired in triplicate. This method assesses theprimary structure of the molecule by acidic hydrolysis of the proteininto its individual amino acid constituents. This method does not detectcysteine or tryptophan. Asparagine and aspartate were detected in asingle peak and labeled Asx. Glutamine and glutamate are also detectedin a single peak and labeled Glx. Of the 20 standard amino acids,fourteen are uniquely detected by this method plus the Asx and Glxgroups for a total of sixteen amino acids. Of those represented, BNJ441has a total of 1262 residues that can be detected by these methods.

Table 19 shows the circular dichroism (CD) Near UV Local Feature, Far UVLocal Feature and Deconvolution results for the BNJ441 batches. Thedeconvolution describes the amounts of α-helix, 3/10 helix, β-sheet,Turns, Poly (Pro) II and unordered structures determined by CDProsoftware against a given reference set. The CD spectra for Near UV(tertiary structure) and for Far UV (secondary structure) for each batchwere determined. This method assessed higher order molecular structure(2° and 3°) in the molecule by the differential absorption of left andright circularly polarized light exhibited in the absorption bands ofoptically active (chiral) molecules, such as proteins. Deconvolution ofthe CD spectra was performed and the results are shown in Table 19.

Table 19 shows the mean molecular weight for each glycan determined. Theobserved N-Linked Oligosaccharide or glycan molecular weights for theBNJ441 batches were consistent with the theoretical glycan molecularweights shown in Table 20. The free glycan molecular weight spectra weredetermined by MALDI-TOF mass spectrometry. This method identified theglycans associated with the drug molecule by molecular weight. Theglycans were previously enzymatically cleaved from the antibody withPNGase F. The glycans were then solid phase extracted and mixed with the3,4-dihydroxybenzoic acid matrix solution and co-precipitated on theMALDI target. This dried sample was ionized with a nitrogen laser into aTOF mass spectrometer. An m/z (M+Na)⁺ spectrum was collected.

TABLE 20 Theoretical Glycan Molecular Weight Glycan TheoreticalStructure m/z (M + Na)+ G1F 1647.58 G1 1501.53 G0F 1485.53 G0 1339.47G0F-GN 1282.45 Man-5 1257.41

The oligosaccharide percentages determined for the BNJ441 batches areshown in Table 19. The totals for various types of N-linkedoligosaccharides were calculated: (Total G0F, G1F), Acidic, HighMannose, Neutral, Monosialylated and Disialylated. The N-linkedoligosaccharides only contained neutral oligosaccharides. The level ofneutral oligosaccharides was 99.99 and 100.0% for the engineering andGMP batches respectively. The oligosaccharides were detected using HPLCand the chromatograms were evaluated quantitatively. This methodevaluates the glycosylation pattern by identifying the N-linkedoligosaccharides associated with the drug molecule on the basis of theretention time of the enzymatically released and fluorescently taggedoligosaccharides. This method provided the relative abundance of eacholigosaccharide species. Briefly, the oligosaccharides wereenzymatically cleaved from the antibody with PNGase F and tagged withanthranilic acid. Excess anthranilic acid was removed using a HILICfiltration step. Samples were injected on to a wAX-HPLC system with aShowa Denko Asahipak Amino Column and the tagged oligosaccharides weredetected with a fluorescence detector; 360 nm excitation and 420 nmemission.

The monosaccharide percentages were determined for the BNJ441 batchesand are shown in Table 19. The monosaccharide percentages were determinefor the five monosaccharides (GlcNAc, GalNAc, Galactose, Mannose,Fucose) using fluorescence labelling followed by reverse phase highpressure chromatography (RP-HPLC). This assay characterizes theglycosylation pattern by determining the monosaccharides associated withthe drug molecule on the basis of the retention time of thefluorescently labelled monosaccharides. Briefly, acid hydrolysis removedthe oligosaccharides from the protein and into its constituentmonosaccharides. The free monosaccharides were then labelled withanthranilic acid (AA) by reductive amination. Samples were then injectedon to an RP-HPLC system with a Waters Symmetry® C-18 column and the AAtagged monosaccharides were detected with a fluorescence detector; 360nm excitation 420 nm emission. Samples were tested in duplicate and thevalue reported was the mean of the two results.

Next we determined the sialic acids N-acetylneuraminic acid (NANA), andN-glycolylneuraminic acid (NGNA). In each case, the determined NANA andNGNA sialic acid content of the BNJ441 batches were below the limit ofquantitation (<6 mmol/mol) as shown in Table 19. No NGNA was observedfor either batch. The sialic acids were measured separated on RP-HPLCfollowing fluorence labelling and using multi-point calibration. Thismethod assesses the glycosylation pattern by determining the type andrelative amount of the sialic acids associated with the drug molecule.The sialic acids were chemically cleaved from the antibody by incubationwith sodium bisulfate then tagged with 0-phenylenediamine. Samples wereinjected on to an RP-HPLC system with a Beckman C18 Ultrasphere columnand the tagged sialic acids were detected with a fluorescence detector(230 nm excitation; 425 nm emission). Samples were tested in duplicatesand the mean of the two results was reported.

The determined T_(m) value of each BNJ441 batch was 67.0° C., as shownin Table 19. Differential scanning calorimetry (DSC) scans wereperformed and calorimetry data acquired using the Micro-Cal VP-DSC byup-scanning at a rate of 75° C./hr from 20° C. to 95° C. The Y-axis andtemperature calibrations were performed prior to sample testing. TheY-axis deflection % error was <1% and transition mid-points were withinthe accepted range of ±0.2° C. of both 28.2° C. and 75.9° C. Sampleswere scanned against blanks of the same buffer composition and volume.DSC measures the enthalpy (AH) of unfolding due to heat denaturation. Abiomolecule in solution is in equilibrium between the native (folded)conformation and its denatured (unfolded) state. The transition midpoint(T_(m)) is the temperature where 50% of the protein is in its nativeconformation and 50% is denatured. The T_(m) for each sample isdetermined by measuring ΔH across a temperature gradient in the samplecell compared to that of the blank cell.

The affinity (K_(D)) for BNJ441 engineering and GMP batch materials were461 pM and 421 pM respectively with good fits. Binding kinetics of eachBNJ441 batch are shown in Table 19. Surface plasmon resonance (Biacore3000) was used to evaluate the binding kinetics of anti-05 antibody(BNJ441) to human C5. Sensorgrams not shown. The kinetics of BNJ441 toC5 were determined using an anti-Fc human capture method. Anti-Fc-Human(KPL #01-10-20) diluted to 0.1 mg/mL in 10 mM sodium acetate pH 5.0 wasimmobilized on two flow cells of a CM5 chip for 8 minutes by aminecoupling. The anti-C5 antibody (BNJ441) was diluted to 0.35 μg/mL inrunning buffer (HBS-EP, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005%P20, pH 7.4). Diluted antibody was then injected on the other flow cell,followed by injections of C5 (0.19-6 nM) on both flow cells. Thesecondary flow cell was used as a reference. The surface was regeneratedeach time with 20 mM HCl, 0.01% P20 (100 μL/min, 200 μL injection). Thedata was processed with a 1:1 Langmuir model using BIAevaluation 4.1with ‘double referencing’.

The affinity (K_(D)) for self association of BNJ441 engineering and GMPbatch materials were 7.1 mM and 0.27 mM respectively. See Table 19. Poorfits were due to low levels of binding observed for both BNJ441engineering and GMP batch materials, self association and the measuredaffinity were below the level of limits of detection of the instrument.A low level of self-association is advantageous for manufacturabilityand ultimately for administration to patients. Sensorgrams not shown.Surface plasmon resonance (Biacore 3000) was used to evaluate theself-association kinetics of anti-C5 antibody (BNJ441). Theself-association kinetics of BNJ441 were determined by directimmobilization of the antibody (BNJ441). BNJ441 was diluted toapproximately 31 μg/mL in 10 mM sodium acetate pH 5.0 was immobilized onone flow cell of a CM5 chip to obtain 2000RU's by amine coupling. Asecondary flow cell was used as a reference. Dilutions of anti-C5antibody, BNJ441 (1.6-50 μM in running buffer, HBS-EP, 0.01 M HEPES,0.15 M NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) was then injected on bothflow cells. No regeneration was necessary due to poor binding. The datawas processed with a steady state affinity model using BIAevaluation 4.1with ‘double referencing’.

The physicochemical characterization of BNJ441 has been conducted usingthe engineering and GMP batches and has been shown to be consistent withthe amino acid sequence for the antibody. The physicochemical datasummarized in this example encompass a range of properties includingpurity, molecular size, identity, structure, glycosylation,thermostability, kinetics and self-association, and are expected toserve as a basis for the characterization of BNJ441 bulk drug substance.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

SEQUENCES REFERENCED IN DISCLOSURE SEQ ID NO: 1 GYIFSNYWIQ SEQ ID NO: 2EILPGSGSTEYTENFKD SEQ ID NO: 3 YFFGSSPNWYFDV SEQ ID NO: 4 GASENIYGALNSEQ ID NO: 5 GATNLAD SEQ ID NO: 6 QNVLNTPLT SEQ ID NO: 7QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGT LVTVSS SEQ IDNO: 8 DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK SEQ ID NO: 9ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 10QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 11DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12 QVQLVQSGAEVKKPGASVKVSCKASGH IFSNYWIQWVRQAPGQGLEWMGEILPGSG HTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQG TLVTVSS SEQID NO: 13 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSV LHEALH S HYTQKSLSLSLGK SEQ ID NO: 14 QVQLVQSGAEVKKPGASVKVSCKASG HIFSNYWIQWVRQAPGQGLEWMGEILPGSG HTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV

HEALH

HYTQKSLSLSLGK SEQ ID NO: 15ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLY I T R E PEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 16:QVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 GASENIYHALN SEQ IDNO: 18 DIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK SEQ ID NO: 19EILPGSGHTEYTENFKD SEQ ID NO: 20 DYKDDDDK SEQ ID NO: 21 HHHHHH SEQ ID NO:22 YPYDVPDYA SEQ ID NO: 23 GHIFSNYWIQ SEQ ID NO: 24 MGWSCIILFLVATATGVHSLEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 25MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26MGWSCIILFLVATATGVHS LEQVQLVQSGAEVKKPGASVKVSCKASGHIFSNYWIQWVRQAPGQGLEWMGEILPGSGHTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 27MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYHALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28MGWSCIILFLVATATGVHS LEQVQLVQSGAEVKKPGASVKVSCKASGYIFSNYWIQWVRQAPGQGLEWMGEILPGSGSTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 29MGWSCIILFLVATATGVHS RDIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 31QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 32NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQHLSHRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 33QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPHDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK SEQ ID NO: 34NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCAQYLSSRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 35QVQLQQPGAELVRPGTSVKLSCKASGYTFTSSWMHWVKQRPGQGLEWIGVIDPSDSYTNYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARGGGSSYNRYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

1-30. (canceled)
 31. A method for treating paroxysmal nocturnalhemoglobinuria (PNH), the method comprising administering to the subjectan antibody, or antigen-binding fragment, thereof in an amount effectiveto treat the C5 mediated complement-associated condition, wherein theantibody, or antigen-binding fragment thereof, binds to complementcomponent human C5, inhibits the cleavage of C5 into fragments C5a andC5b, has a serum half-life in humans of at least 40 days, and comprises:(i) a heavy chain CDR1 comprising the amino acid sequence depicted inSEQ ID NO:23, a heavy chain CDR2 comprising the amino acid sequencedepicted in SEQ ID NO:19, a heavy chain CDR3 comprising the amino acidsequence depicted in SEQ ID NO:3, a light chain CDR1 comprising theamino acid sequence depicted in SEQ ID NO:4, a light chain CDR2comprising the amino acid sequence depicted in SEQ ID NO:5, and a lightchain CDR3 comprising the amino acid sequence depicted in SEQ ID NO:6;and (ii) a variant human IgG Fc constant region that binds to humanneonatal Fc receptor (FcRn), wherein the CH3 domain of the variant humanFc constant region comprises Met-429-Leu and Asn-435-Ser substitutionsat residues corresponding to methionine 428 and asparagine 434 of anative human IgG Fc constant region, each in EU numbering.
 32. Themethod of claim 31, wherein the antibody, or antigen-binding fragmentthereof, comprises a heavy chain variable region depicted in SEQ IDNO:12 and a light chain variable region depicted in SEQ ID NO:8.
 33. Themethod of claim 31, wherein the antibody, or antigen-binding fragmentthereof, further comprises a heavy chain constant region depicted in SEQID NO:13.
 34. The method of claim 31, wherein the antibody, orantigen-binding fragment thereof, comprises a heavy chain polypeptidecomprising the amino acid sequence depicted in SEQ ID NO: 14 and a lightchain polypeptide comprising the amino acid sequence depicted in SEQ IDNO:
 11. 35. The method of claim 31, wherein the isolated antibody, orantigen-binding fragment thereof, binds to human C5 at pH 7.4 and 25° C.with an affinity dissociation constant (K_(D)) that is in the range 0.1nM≦K_(D)≦1 nM.
 36. The method of claim 31, wherein the antibody, orantigen-binding fragment thereof, binds to human C5 at pH 6.0 and 25° C.with a K_(D)≧10 nM.
 37. The method of claim 31, wherein the [(K_(D) ofthe antibody, or antigen-binding fragment thereof, for human C5 at pH6.0 and at 25° C.)/(K_(D) of the antibody, or antigen-binding fragmentthereof, for human C5 at pH 7.4 and at 25° C.)] is greater than 25.